Multidrug resistance-associated polypeptide

ABSTRACT

Compositions and methods are disclosed for improving the effectiveness of a chemotherapeutic regimen to eradicate multidrug-resistant transformed cells from the body of a mammal, preferably from the body of a human. The present disclosure capitalizes on the discovery of a novel multidrug-resistance associated protein (MRP), herein designated MRP-β. The disclosed compositions include MRP-β nucleic acids, including probes and antisense oligonucleotides, MRP-β polypeptides and antibodies, MRP-β expressing host cells, and non-human mammals transgenic or nullizygous for MRP-β. The disclosed methods include methods for attenuating aberrant MRP-β gene expression, protein production and/or protein function. In addition, methods are disclosed for identifying and using a modulator, such as an inhibitor, of MRP-β. Preferably, the modulator is a small molecule.

This patent application is a continuation application of U.S. patentapplication Ser. No. 09/061,400, filed on Apr. 16, 1998 (U.S. Pat. No.6,077,936), which in turn is a continuation-in-part of U.S. Ser. No.08/843,459, filed Apr. 16, 1997 (U.S. Pat. No. 6,162,616), thedisclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to cancer chemotherapy. Theinvention relates more specifically to compositions and methods forimproving the effectiveness of a chemotherapeutic regimen to eradicatemultidrug-resistant transformed cells from the body of a mammal,preferably from the body of a human. In this regard, the inventioncapitalizes on the discovery of a novel multidrug-resistance associatedpolypeptide (MRP), herein designated MRP-β. The invention furtherrelates to drug discovery, especially to the design of novelchemotherapeutic drugs that are cytotoxic to cells expressing MRP-β.

BACKGROUND OF THE INVENTION

Cancer chemotherapy involves the administration of one or more cytotoxicor cytostatic drugs to a cancer sufferer. The goal of chemotherapy is toeradicate a substantially clonal population (colony) of transformedcells from the body of the individual, or to suppress or to attenuategrowth of the colony, which is most commonly referred to as a tumor.Tumors may occur in solid or liquid form, the latter comprising a cellsuspension in blood or another body fluid. A secondary goal ofchemotherapy is stabilizaon (clinical management) of the afflictedindividual's health status. Although the tumor may initially respond tochemotherapy by, e.g., stabilizing or reducing its growth rate, in manyinstances the initial chemotherapeutic treatment regimen becomes lesseffective or ceases to impede tumor growth. Conventional treatmentregimes endorse the use of additional or substitute chemotherapeuticdrugs, including drug combinations, in an effort to regain control overtumor growth. However, it is well known that transformed cells in atumor may acquire resistance to a broad spectrum of chemotherapeuticdrugs, including drugs to which the tumor has not hitherto been exposedduring treatment. This acquisition of a multidrug-resistant (ormultidrug-resistance) phenotype significantly constrains thechemotherapeutic choices available to the clinician, and significantlyworsens prognosis for the afflicted individual. Acquisition of multidrugresistance is particularly problematic in carcinomas originating insecretory epithelia, including lung, gastrointestinal tract, mammary,reproductive tract, endocrine and neuroendocrine epithelia.

Tumor cell transformation is the process by which a cell escapes normalcontrol mechanisms governing the cell's tissue-specific phenotype anddifferentiation state. Thus, transformation often involves“dedifferentiation,” which is defined as an inappropriate return to aless committed or less tissue-specific phenotype. Alternatively,transformation involves incomplete or arrested differentiation of cellsnormally responsible for replenishing cells lost to normal tissueturnover. Transformed cells of epithelial origin produce tumors that arecarcinoma cell colonies (carcinomas). When in a gland-like configurationor derived from secretory tissue, such epithelium-derived tumors arereferred to as adenocarcinomas. In contrast, transformed cells ofmesenchymal origin produce tumors that are sarcoma cell colonies(sarcomas). Transformed cells of the hematopoietic lineage produceleukemias, lymphomas or lymphosarcomas, each of which often occur ascell suspension tumors. In contrast, the primary tumor growth of acarcinoma or sarcoma usually remains near the site of initial celltransformation. However, secondary foci (metastases) of tumor growth canarise at other sites, which can be far removed from the primary tumorgrowth site. The presence and/or abundance of metastases indicates thedegree to which transformed cells have strayed from their normaltissue-specific phenotype and/or acquired invasive properties.

Phenotypically, cell transformation involves the display of altered orabnormal structural (e.g. antigenic) and functional cellular properties.These altered properties provide the transformed cell with a survival orgrowth advantage over neighboring, non-transformed cells in its tissueof origin. The advantage may arise from acquisition of autocrine growthregulation, abnormal activation of genes controlling or regulating thecell division cycle, abnormal suppression of genes needed for normalexit from or arrest of the cell division cycle, or other changesaffecting cell growth and/or survival. Over time, divisions of thetransformed cell produce a colony (tumor) of daughter cells each havingthe phenotypic advantage gained by the original transformed cell. Theimposition of chemotherapy subjects the tumor to selection pressure, ineffect encouraging further phenotypic change by which tumor cells mayescape the cytotoxic effects of a chemotherapeutic drug. Thus, thestructural and functional properties of transformed cells in a tumor canfluctuate over time and over the course of chemotherapeutic treatment.

A significant survival advantage is associated with the acquisition of amultidrug-resistance phenotype, which arises from expression of acellular gene encoding a protein that removes diverse chemotherapeuticdrugs or drug metabolites from the intracellular milieu. Drug exportdiminishes cytotoxic effect, thereby protecting the transformed cellfrom otherwise lethal chemotherapeutic drugs or drug concentrations. Todate, two genes encoding multidrug-resistance export proteins have beenidentified in the human genome. The first of these, MDR1, encodesP-glycoprotein, a 170 kDa multispanning transmembrane protein belongingthe ATP Binding Cassette (ABC) Transporter protein superfamily. Lautieret al. (1996), 52 Biochem. Pharmacol 967-977. Superfamily members aremultispanning transmembrane proteins that transport substances into orout of the intracellular environment in an energy-dependent manner.Higgins (1992), 8 Ann. Rev. Cell Biol. 67-113, provides a generaloverview of the properties and natural occurrence of superfamily memberproteins. ABC transporters have been identified for a large variety ofstructurally diverse transported substrates, including sugars, peptides,inorganic ions, amino acids, polysaccharides and proteins. Individualtransporter proteins appear to function unidirectionally, i.e., to carryout either export or import of intracellular substances. Thus,P-glycoprotein functions by exporting chemotherapeutic drugs which,although structurally heterogenous, appear to share hydrophobicproperties. P-glycoprotein overexpression correlates with the presenceof a multidrug-resistance phenotype in diverse tumor cell isolates andtumorigenic cell lines. Significant effort has been invested in thedevelopment of agents to block or attenuate P-glycoprotein mediated drugexport. Such agents are referred to commonly as “chemosensitizers” or“MDR reversal agents,” and are disclosed in Hait et al. (1992), U.S.Pat. No. 5,104,858; Sunkara et al. (1993), U.S. Pat. No. 5,182,293;Sunkara et al. (1993), U.S. Pat. No. 5,190,957; Ramu et al. (1993), U.S.Pat. No. 5,190,946; Powell et al. (1995), U.S. Pat. No. 5,387,685;Piwnica-Worms (1995), U.S. Pat. No. 5,403,574; Sarkadi et al. (1995),PCT Publication WO 95/31474; Sunkara et al. (1996), U.S. Pat. No.5,523,304; Zelle et al. (1996), U.S. Pat. No. 5,543,423; Engel et al.(1996), U.S. Pat. No. 5,556,856; Powell et al. (1996), U.S. Pat. No.5,550,149 and Powell et al. (1996), U.S. Pat. No. 5,561,141. However,P-glycoprotein overexpression does not account for all instances of theacquisition of a multidrug-resistance phenotype. Lautier et al. (1996),52 Biochem, Pharmacol. 967-977.

A second multidrug-resistance gene identified to date in the humangenome encodes multidrug-resistance associated protein (MRP), a 190 kDamultispanning transmembrane protein also belonging to the ABCTransporter protein superfamily. MRP is described in Deeley et al.(1996), U.S. Pat. No. 5,489,519, the teachings of which are incorporatedby reference herein. MRP shares only 15% sequence identity withP-glycoprotein at the amino acid level. In addition, MRP differs fromP-glycoprotein in its ability to expel specific types ofchemotherapeutic drugs from the intracellular milieu. These differencesare thought to arise from differences in the drug expulsion mechanism ofthe two proteins: MRP appears to act on a glutathione-derivatized drugmetabolite, whereas P-glycoprotein appears to act on an underivatizeddrug. Lautier et al. (1996), 52 Biochem. Pharmacol. 967-977.Significantly, agents that block or interfere with P-glycoproteinfunction appear to have little crossreactivity with MRP. Thus,significant effort is being invested in the development of substances(MDR reversal agents) that block or inhibit MRP function.

Overexpression of either P-glycoprotein or MRP can endow a transformedcell with a multidrug-resistance phenotype; thus, empirical testing isrequired to determine whether a particular reversal agent will beeffective for interfering with a tumor's multidrug resistance phenotype.Currently, it is unclear whether MRP and/or P-glycoprotein expressionaccounts for all occurrences of the multidrug-resistance phenotype,which arises fairly commonly during the course of chemotherapeutictreatment, irrespective of the tissue specificity of the primary tumor.Moreover, the expression patterns of MRP and P-glycoprotein within agiven cell population have been observed to fluctuate over time. Thus,exposure to a reversal agent that interferes with P-glycoproteinfunction may impose selection pressure favoring the expression of MRP.Such pressure would result in continued viability of cells having amultidrug resistance phenotype. Lautier et al. (1996), 52 Biochem.Pharmacol 967-977.

Needs remain for preventing or reversing the acquisition of a multidrugresistance phenotype in transformed cells. Particular needs remain toestablish the mechanism(s) by which the multidrug resistance phenotypecan be produced, and to provide additional therapies for restoring drugsensitivity to multidrug-resistant transformed cells. Still moreparticular needs remain to improve the clinical management of multidrugresistant tumors, especially when the multidrug resistance phenotypearises entirely or partially from overexpression of one or more genesother than those encoding P-glycoprotein or MRP.

SUMMARY OF THE INVENTION

The present invention capitalizes on the unexpected discovery of a novelgene encoding a hitherto-unknown multidrug-resistance associatedpolypeptide (MRP). This novel polypeptide, designated herein as MRP-β,is encoded in the human genome and is expected to be found in thegenomes of additional mammals. MRP-β likely is a transmembrane-spanning,energy-dependent transporter or pump, as are other members of the ATPBinding Cassette (ABC) Transporter Protein superfamily to which theknown proteins MRP and P-glycoprotein belong. It is likely that MRP-β isdisposed in the plasma membrane of a mammalian cell, and functions byejecting intracellular substances, such as chemotherapeutic drugs.Alternatively, MRP-β may span a vesicular membrane, and function bysequestering intracellular substances. Elevated levels of expression ofthe novel MRP-β gene, or of bioactivity of the novel MRP-β polypeptideencoded by this gene, accordingly are expected to contribute to theemergence and/or persistence of a multidrug-resistance phenotype intransformed mammalian cells, such as carcinoma cells, includingadenocarcinoma cells. Elevated expression or bioactivity of MRP-βsimilarly is expected to contribute to the occurrence of amultidrug-resistance phenotype in sarcoma cells and in transformed cellsof the hematopoietic lineage, including leukemias, lymphomas andlymphosarcomas. MRP-β is likely to account for multidrug-resistantmammalian cell phenotypes that are refractory to treatment with reversalagents that interfere with expression, production and/or function ofP-glycoprotein or of MRP.

Accordingly, it is an object of this invention to provide nucleic acidsand expression vectors encoding MRP-β or a unique fragment thereof. Itis another object to provide nucleic acids, including probes andantisense oligonucleotides, complementary to MRP-β encoding nucleicacids. An additional object is to provide methods and compositions formitigating aberrant expression of an MRP-β gene, or for mitigatingaberrant bioactivity of an MRP-β polypeptide. It is yet another objectto provide methods and compositions for characterizing and/orattenuating a multidrug resistance phenotype. It is still another objectto provide methods and compositions, including MRP-β expressing hostcells, for identifying one or more modulators, preferably inhibitors, ofMRP-β. A still further object includes the modulation, preferably theinhibition, of MRP-β and of disease states associated with MRP-β. A yetfurther object includes the potentiation of chemotherapy to eradicatemultidrug resistant transformed cells from the body of an individual,such as a cancer patient. These and other objects, along with advantagesand features of the invention disclosed herein, will be apparent fromthe description, drawings and claims that follow.

In a first aspect, the invention features nucleic acids encoding orcomplementary to MRP-β or a unique fragment thereof. A preferredembodiment provides nucleic acid, the sequence of which comprises SEQ IDNo: 1, an MRP-βcDNA sequence. Another preferred embodiment providedMRP-β cDNA deposited on Apr. 16, 1997, under the terms of the BudapestTreaty with the Culture Collection (ATCC), 10801 University BoulevardManassas, Va. 20110-2209. The deposited cDNA is herein designatedfohd013a05m and is accorded Deposit No. 9409. Another preferredembodiment provides ribonucleic acid (RNA) encoding an MRP-βpolypeptide, the amino acid sequence of which comprises SEQ ID No: 2.Messenger RNA (mRNA) encoding MRP-β is approximately 6 kilobases (kb) inlength. Other embodiments provide unique fragments (e.g., SEQ ID No: 3)of the MRP-β cDNA, including fragments corresponding to portions of theopen-reading frame (ORF), and fragments corresponding to untranslatedsequences 3′ or 5′ to the ORF. These unique fragments can be used toproduce or design probes for the analysis of cellular MRP-βexpressionpatterns, e.g., for purposes of diagnosing an abnormality in orcontributed to by MRP-β. In addition, the present fragments can be usedfor the production or design of polymerase chain reaction (PCR) primersor antisense oligonucleotides, including therapeutic oligonucleotidesthat disrupt cellular MRP-β gene expression, especially abnormal oraberrant expression. It will be understood that the present nucleicacids, especially probes and oligonucleotides, may be detectablylabelled and/or may comprise one or more modifications in a nucleotidebase, backbone sugar or phosphate, or be linked together by linkagesother than phosphodiester bonds.

The invention is further embodied in nucleic acids that hybridize to SEQID No: 1 or to the complement thereof. Preferred nucleic acids hybridizeto SEQ ID No: 1 or to the complement thereof under stringent conditions.Preferred antisense and/or primer oligonucleotides hybridize to uniquefragments of SEQ ID No: 1 or of the complement thereof, e.g., underintracellular conditions. Additional MRP-β variant nucleic acidsprovided herein comprise nucleotide sequences at least 50%, preferably60%, 70%, 80%, more preferably 90% and even more preferably 95%identical to SEQ ID No: 1. The present variant nucleic acids comprisenucleotide mutations (substitutions, deletions and/or insertions)distributed in any random or non-random frequency within the SEQ ID No:1 sequence. The invention further provides degenerate variant nucleicacids that encode the SEQ ID No: 2 polypeptide or a unique fragmentthereof. In yet further embodiments, the invention provides nucleicacids encoding variant MRP-β polypeptides, comprising amino acidsequences sharing at least 75% sequence similarity with the SEQ ID No: 2polypeptide. Preferably, these nucleic acids encode polypeptides sharingat least 80%, 85%, 90% or more preferably 95% amino acid sequencesimilarity with the SEQ ID No: 2 MRP-β polypeptide. The encoded variantpolypeptides comprise amino acid mutations (substitutions, deletionsand/or insertions) distributed in any random or non-random frequencywithin the SEQ ID No: 2 sequence. “Similarity” as used herein refers tothe sum of aligned amino acid residues that are identical to thecorresponding SEQ ID No: 2 residues and those that are allowed pointmutations therefor. Moderate gaps and/or insertions (e.g., less thanabout 50, preferably less than about 15, more preferably less than about5 amino acid residues) in the aligned sequence are ignored forsimilarity calculation purposes. Allowed point mutations aresubstitutions by amino acid residues that are physically and/orfunctionally similar to the corresponding aligned SEQ ID No: 2 residues,e.g., that have similar size, shape, hydrophilic or hydrophobiccharacter, charge and chemical properties.

It should be understood that the present invention providesoligonucleotides that hybridize to any of the foregoing variant MRP-βnucleic acids, i.e., to nucleic acids that encode polypeptidescomprising amino acid sequences that share at least 75% sequencesimilarity with the SEQ ID No: 2 polypeptide. More particularly, theinvention provides olignucleotides that hybridize to one or more uniquefragments of nucleic acids encoding the present MRP-β polypeptides. Fortherapeutic purposes and/or for PCR investigative or diagnosticpurposes, the present oligonucleotides hybridize to a unique fragmentcomprising 5′ untranslated sequence, a transcription initiation site,ORF or polypeptide coding sequence, intron-exon boundary,polyadenylation site or 3′ untranslated region of the present MRP-βnucleic acids. Exemplary antisense oligonucleotides are disclosed herein(SEQ ID Nos: 4, 5, 6, 7 and 8).

For antisense-oligonucleotide based therapeutic purposes, one or more ofthe present antisense MRP-β oligonucleotides (optionally comprising oneor more modified moieties as disclosed herein) is formulated togetherwith a pharmaceutically acceptable vehicle to produce an antisensepharmaceutical composition suitable for local or systemic administrationto a mammal, or for treatment of mammalian cells or tissue whether insitu or ex vivo. In an alternative embodiment, the present antisenseoligonucleotide is encoded by an antisense expression vector comprisinga nucleic acid insert complementary to the oligonucleotide sequence. Theantisense vector preferably comprises or is packaged with one or moreretroviral elements for infection of mammalian cells, and furthercomprises one or more conventional expression control elements (e.g., apromoter, transcriptional initiation site, termination site, or thelike) to direct intracellular production of the antisenseoligonucleotide in infected cells. The present vector also can beformulated with a pharmaceutically acceptable vehicle to produceadditional antisense pharmaceutical compositions of the presentinvention. Thus, the antisense vector, when internalized by a cell(e.g., by retroviral infection, pinocytosis or diffusion), directs theintracellular production of an antisense oligonucleotide which, as doany of the therapeutic antisense oligonucleotides disclosed herein,disrupts cellular expression of an MRP-β gene. Disruption of expressionis achieved by interfering with MRP-β gene activation or transcription,by destabilization of MRP-β gene transcripts, or by interference withthe translation of MRP-β gene transcripts. In this manner, the presentinvention provides compositions for mitigating aberrant expression of anMRP-β gene, e.g., expression which contributes to the emergence orpersistence of a multidrug-resistant phenotype.

In a second aspect, the invention features an MRP-β polypeptide, theamino acid sequence of which comprises SEQ ID No: 2. More generally, theinvention provides MRP-β polypeptides, and unique fragments (epitopes)thereof, that are encoded by any of the above-described MRP-β nucleicacids. For example, the invention provides MRP-5 polypeptides, the aminoacid sequences of which comprise a sequence sharing at least 75%sequence similarity (as defined herein) with SEQ ID No: 2. Such MRP-βpolypeptides include naturally-occurring variants (e.g., polymorphicvariants, phylogenetic counterparts of the presently disclosed humanMRP-β, and/or naturally-occurring mutant variants, particularly mutantsassociated with the process of somatic cell transformation ortumorigenesis) and biosynthetic variants produced by routine molecularengineering techniques. Based upon an assessment of its sequencesimilarity to known proteins, such as MRP, the present novel MRP-Dpolypeptide is believed to be a novel member of the ABC TransporterProtein superfamily. Thus, it is anticipated that MRP-β polypeptideswill be displayed on the surface of cells expressing an MRP-βgene, suchas multidrug resistant tumor cells or transfected host cells. Of course,it is also possible that MRP-β will be incorporated into intracellularphospholipid membranes, such as vesicular membranes. Cellular productionof MRP-β is expected to contribute to the emergence and/or persistenceof a multidrug-resistant phenotype in transformed mammalian cells. Thepresent invention provides various specific MRP-βpolypeptideembodiments, including MRP-β polypeptides immunogenically displayed onintact host cell membranes or cell-free membrane fractions derived fromhost cells; MRP-β polypeptides incorporated into synthetic ornon-cellular phospholipid membranes or micelles, and MRP-β polypeptidesand polypeptide fragments isolated in substantially pure form. Any ofthe foregoing polypeptides, or unique, immunogenic fragments (epitopes)thereof can be used to induce immune responses in human or nonhumanmammals.

Accordingly, in a third aspect, the invention features an antibody thatbinds selectively to an epitope unique to MRP-β. Preferably, theinvention provides an antibody that binds to an MRP-β epitope that isdisplayed on the surface of MRP-βexpressing cells, such as transformedor host cells. Antigen-binding fragments of the present antibody alsoare provided herein. Such fragments include truncated forms of theantibody that retain antigen binding properties, e.g., Fab, Fab₂, Fab′and Fv fragments thereof. Such fragments are produced conventionally byenzymatic or chemical cleavage of an intact antibody of the presentinvention. Alternatively, such fragments can be produced throughmolecular engineering techniques. In certain embodiments, the presentantigen binding fragment is incorporated into a fusion polypeptide, suchthat the fragment is fused to another polypeptide, such as animmunoglobulin framework polypeptide. An exemplary framework is of humanorigin. Alternatively, the antigen-binding region is fused to anon-immunoglobulin polypeptide, e.g., to a cytotoxin or to achemoattractant polypeptide. A cytotoxin polypeptide induces or mediatescell death (cytolysis), e.g., by inducing apoptosis or by disruptingcell metabolism, cell membrane integrity, intracellular fluid volume, orthe like. Exemplary cytotoxic fusion proteins comprise ricin, diptheriatoxin, or another naturally-sourced toxin of plant, animal or microbialorigin. A chemoattractant polypeptide is any polypeptide of mammalianorigin that induces or stimulates activation and localization of immuneeffector cells (e.g., natural killer cells, cytotoxic T cells,macrophages and the like) that typically mediate a cellularproinflammatory immune response. Exemplary chemoattractant fusionproteins comprise a chemokine, lymphokine or cytokine polypeptide (e.g.,interleukin-2 (IL2), tumor necrosis factor TNF), and the like).

In a fourth aspect, the invention features expression vectors comprisingnucleic acid encoding an MRP-β polypeptide comprising an amino acidsequence that shares at least 75% sequence similarity with SEQ ID No: 2.The nucleic acid sequence of an exemplary expression vector thuscomprises SEQ ID No: 1. The nucleic acid sequence of another exemplaryexpression vector comprises the sequence of MRP-βcDNA deposited on evendate herewith. Additional exemplary expression vectors comprise nucleicacid encoding variants, whether biosynthetic or naturally-sourced, ofthe presently disclosed MRP-β polypeptide. Certain embodiments of thepresent expression vectors encode chimeric polypeptides in which one ormore MRP-β amino acid residues are substituted by the correspondingresidues of another ABC Transporter Protein superfamily member, such asMRP or P-glycoprotein. Such embodiments are expected to facilitateelucidation of the molecular basis of multidrug resistance phenotypes,and thence to facilitate design or screening of novel inhibitors ofmultidrug resistance. In addition to nucleic acid encoding the MRP-βpolypeptide, the present expression vectors comprise one or moreexpression control elements (e.g., promoter, transcriptional initiationsite, termination site and the like) to direct the production of theencoded MRP-β polypeptide in prokaryotic or, preferably eukaryotic, hostcells. Optionally, the present expression vectors further comprise aselectable marker gene. For use with eukaryotic host cells, the presentexpression vector may still further comprise one or more retroviralcomponents to promote infectivity and uptake by eukaryotic, preferablymammalian, cells.

Accordingly, a fifth aspect of the present invention features a hostcell transfected with an above-described expression vector. A preferredhost cell displays a vector-encoded MRP-β polypeptide, comprising asequence sharing at least 75% sequence similarity with SEQ ID No: 2, onthe cell surface. A particularly preferred host cell displays afunctional and immunologically detectable MRP-β polypeptide. In otherembodiments the vector encoded MRP-β polypeptide may reside within thecell, e.g., as a component of a vesicular membrane. Preferred host cellsacquire a multidrug resistance phenotype and are able to eject orsequester intracellular substances, including chemotherapeutic drugsand/or metabolites thereof. The present host cells can be of human ornon-human origin, and can be naturally-sourced, adapted to primaryculture, or immortalized under culture conditions. Cells that aresuitable for production of host cells are herein defined as sourcecells. Exemplary source cells include normal differentiated mammaliancells (e.g., obtained by biopsy), cells in primary culture (e.g.,serially passaged benign or malignant transformed cells), and cell lines(e.g., immortalized transformed cells such as HeLa, MCF-7, and thelike). Preferably, mammalian source cells are primate cells, mostpreferably human cells. For screening or other investigative purposes,such as the production of non-human mammals, rodent, ovine, porcine,bovine or other mammalian source cells may be used. In otherembodiments, host cells can be produced from prokaryotic or eukaryoticsource cells, e.g., unicellular organisms, such as yeast. Any of theforegoing can be used to produce host cells by standard celltransfection or infection techniques. Thus, an MRP-β expression vectorcan be stably incorporated into a source cell by transfection,pinocytosis, electroporation, microinjection, retroviral infection orthe like. Transfected cells then are cultured under conditions favorableto the selective survival of MRP-βexpressing host cells, e.g., in thepresence of a drug cytotoxic to source cells but to which expression ofMRP-β or a vector-borne selectable marker gene confers a survivaladvantage for host cells. Host cells so obtained are useful for theproduction and characterization of MRP-β antibodies, for investigationof the nature and variety of toxic substances subject to MRP-βtransport, and for the screening and identification of MRP-β inhibitorsas described herein.

In further embodiments, MRP-β host cells can be produced fromuncommitted source cells, preferably embryonic stem cells or blastocystcells, of non-human mammalian origin. An uncommitted cell is one that iscompetent to differentiate, under appropriate conditions, intodifferentiated cells of one or more specific mammalian body tissues. Inthe present embodiment, an MRP-β expression vector is introduced into anuncommitted embryonic source cell and preferably integrates in asite-specific or nonspecific fashion into the cells' genome to produce ahost cell competent to differentiate into one or a plurality ofdifferentiated cell types. Alternatively, the present expression vectorresides in the host cell as microsatellite DNA. In some embodiments, thepresent expression vector confers a tissue-specific pattern of MRP-βexpression in tissue arising from the differentiation of uncommittedhost cells. Uncommitted host cells can, through manipulation byestablished techniques, be used to produce non-human mammals that areeither transgenic or nullizygous for MRP-β. To produce a transgenicmammal of the present invention, an above-described host embryonic stemcell or blastocyst cell is integrated (e.g., by microinjection) into anon-human mammalian blastocyst, which is thereafter implanted into theuterus of a non-human, pseudopregnant mammal, such as a mouse, rat,rabbit, sheep, goat, pig or cow. Following a normal gestation period,this intrauterine implantation procedure yields a non-human foundermammal, the body tissues of which comprise a mosaic of normal cells andhost cells, the latter comprising MRP-β nucleic acid of vector origin.Progeny of the present founder mammal are characterized by germlineintegration of nucleic acid of vector origin. Transgenic progeny expressan MRP-β polypeptide, the amino acid sequence of which comprises asequence sharing at least 75% sequence similarity with SEQ ID No: 2.Optionally, this polypeptide is expressed in a tissue-specific manner.Thus, transgenic progeny constitutively or inducibly express MRP-β inall or a subset of their body tissues. Cells isolated or, optionallyimmortalized from, such transgenic tissue are expected to facilitateinvestigations into the discovery and characterization of MRP-βmodulators useful for treatment of multidrug-resistant transformed cellsarising in any mammalian body tissue. For example, transgenic progenyand/or their cells can be used to confirm whether substances initiallyidentified as modulators in an in vitro screen suppress MRP-βpolypeptide production or biological function in vitro. Advantageously,transgenic progeny provide a tissue source that can be matched to atissue type for which modulators of multidrug resistance areparticularly desired, e.g., which has a known propensity for developingmultidrug resistance. Such tissue types include, but are not limited to,mammary, respiratory tract, gastrointestinal tract, urogenital tract,hematopoietic and endocrine system tissue.

To produce a nullizygous mammal of the present invention, anabove-described uncommitted source cell is transfected (e.g., infected)with a null vector, which comprises a non-expressible variant of theMRP-β encoding nucleic acid disclosed herein. The null vector furthercomprises sufficient nucleic acid sequence 5′ and 3′ to the MRP-β ORF toachieve homologous recombination with any endogenous MRP-β gene presentin the source cells' genome. As a result of homologous recombination,any endogenous MRP-β gene is nullified, i.e., replaced by the presentnon-expressible variant. Appropriate non-expressible variants includeantisense-oriented MRP-β nucleic acids, nucleic acids comprisingpremature stop codons in the ORF, nucleic acids comprising a defectivepromoter, and the like. The present null host cell is integrated into ablastocyst and implanted into a pseudopregnant mannal to produce a nullfounder mammal. Progeny of this founder are characterized by gemnlineintegration of nucleic acid derived from the null vector. Thus, innullizygous progeny, the ability to express a naturally encoded MRP-βhomolog is “knocked out” such that, preferably, the progeny areincapable of developing a multidrug resistance phenotype attributable toMRP-β expression. Such nullizygous progeny and/or their cells can beused to assess potential side effects or undesirable consequences ofMRP-β modulator (e.g., inhibitor) therapy. Nullizygous progeny and/ortheir cells also can be used to detect additional genes that contributeto emergence of a multidrug-resistance phenotype, i.e., genes other thanMRP-β, MRP and P-glycoprotein. Cells isolated or cultured fromnullizygous progeny can be exposed to selection pressure by culturingthem in the presence of a chemotherapeutic drug, and monitoring thecultures for emergence of a drug-resistant phenotype. Optionally, theMRP-β nullizygous progeny provided herein can be crossbred withnon-human mammals nullizygous for MRP nd/or P-glycoprotein. Suchmultiply nullizygous progeny should facilitate screening for additionalgenes that can contribute to the emergence of a multidrug resistancephenotype.

The above-described MRP-β compositions are useful according to teachingsherein for assessing the presence of mutations in an MRP-β gene;assessing MRP-β gene expression level, especially for detectingfluctuations in expression; and, for mitigating aberrant expressionand/or biological function of an MRP-β polypeptide. Preferably, thepresent MRP-β compositions are useful to treat a disease state or otherdeleterious condition contributed to by aberrant MRP-5 gene expressionor biological function. Most preferably, the present MRP-β compositionsare useful to attenuate and/or to abrogate a multidrug resistantphenotype, e.g., of transformed cells in the body of a cancer sufferer.As a result, the present invention offers means for potentiatingchemotherapy to eradicate multidrug-resistant transformed cells from anindividual's body.

Thus, in a sixth aspect, the invention features diagnostic methods fordetecting abnormalities in an MRP-5 gene. In one embodiment, theinvention provides a method of detecting a mutation or other structuralabnormality in an MRP-β gene. Mutations, whether of germline or somaticorigin, may indicate whether the process of cell transformation(tumorigenesis) has been initiated or is likely to arise in anindividual's tissues. Mutations are detected by obtaining cellulartissue from a mammal, preferably a human, suspected of harboring avariant MRP-β gene, and treating the tissue so as to release nucleicacids therefrom. Preferably the cellular tissue is obtained from a bodytissue suspected of comprising transformed cells. Thus, the presentmethod provides information relevant to diagnosis of the presence of atumor. The method may be practiced with any body tissue type whichcomprises cells, including body fluid cell suspensions (e.g., blood,lymph, cerebrospinal fluid, peritoneal fluid or ascites fluid). Releasedcellular nucleic acids are combined, under hybridization conditions,with an oligoucleotide of the present invention, e.g., anoligonucleotide complementary to nucleic acid encoding MRP-β.Preferably, the oligonucleotide is complementary to a unique fragment ofthe full-length MRP-β nucleic acid. Following incubation with theoligonucleotide under suitable hybridization conditions, the releasednucleic acids are assayed for formation of a hybrid comprising theoligonucleotide. In a preferred embodiment wherein the oligonucleotideis complementary to SEQ ID No: 1 or a unique fragment thereof, formationof the hybrid confirms that the individual harbors at least onewild-type MRP-β gene allele (comprising SEQ ID No: 1). Failure to form ahybrid under stringency conditions that do not tolerate base pairmismatching confirms that the individual lacks a wild-type allele, i.e.,that the individual harbors an aberrant, e.g., mutant, variant of theMRP-β gene.

In another embodiment, the invention provides a method of assessingexpression, especially aberrant expression, of a cellular MRP-β gene. Aswith the preceding embodiment, aberrant expression may indicate thepresence, persistence or reappearance of multidrug-resistant tumor cellsin an individual's tissue. More generally, aberrant expression mayindicate the occurrence of a deleterious or disease-associated phenotypecontributed to by MRP-β. MRP-β gene expression is assessed by obtaininga sample of cellular tissue from a mammal (e.g., a human), preferablyfrom a body site implicated in a possible diagnosis of diseased ormalignant tissue, and treating the tissue to release RNA therefrom.Cellular RNA is combined with an MRP-βoligonucleotide generally asdescribed above, and the resulting mixture is assayed for the presenceof a hybrid comprising the MRP-β oligonucleotide and a cellular MRP-βgene transcript. In preferred embodiments, the presence and/or relativeabundance of this hybrid is expected to indicate aberrant expression ofa cellular MRP-β gene, and to correlate with the occurrence in situ oftransformed cells, especially transformed cells having amultidrug-resistant phenotype.

Preferably, the foregoing embodiments can be practiced using adetectably labeled or otherwise modified MRP-β oligonucleotide, mostpreferably with an oligonucleotide comprising a peptide-nucleic acidbackbone.

In yet another embodiment, the invention provides a diagnostic methodusing an above-described antibody or fragment thereof to characterizeaberrant MRP-βassociated phenotype, e.g., drug-resistant phenotype of atransformed cell. This method involves obtaining cellular tissue from amammal (e.g., a human) suspected of harboring transformed cells, andcontacting the tissue with an above-described antibody under conditionssuch that, if cells of the obtained tissue display a recognized epitopeunique to MRP-β, an antibody-epitope complex forms. Generally, themethod is practiced with intact cells. The practitioner may, however,desire to generate a more sensitive assay for total cellular MRP-βcontent. In these circumstances, the method is practiced withpermeabilized or solubilized cells, which can be produced by exposingthe cells to heat, mechanical disruption, detergent, hypo- orhyper-osmotic conditions, and like conventional techniques. After asufficient period of time has elapsed for formation of theantibody-epitope complex, the tissue is assayed for presence of thecomplex, formation or abnormal elevation of which indicates presence inthe tissue of cells abnormally expressing MRP-β. As disclosed herein,such cells are likely transformed cells characterized by adrug-resistance phenotype.

Information obtained from practice of the foregoing diagnostic methodsis expected to be useful in prognostication, staging and clinicalmanagement of diseases and other deleterious conditions affecting anindividual's health status. In preferred embodiments, the foregoingdiagnostic methods provide information useful in prognostication,staging and management of malignancies (tumors) that are characterizedby expression of MRP-β and thus by a multidrug-resistance phenotype. Theinformation more specifically assists the clinician in designingchemotherapeutic or other treatment regimes to eradicate suchmalignancies from the body of an afflicted mammal, typically a human.The present methods can be practiced with any samples of any body tissuetype, and are desirable for assessing cellular tissue of mammary,respiratory tract, urogenital tract, endocrine system or immune systemorigin. The present methods are particularly useful to assess breastbiopsy, bronchoalveolar lavage, ovarian, uterine or cervical biopsy,prostate or testicular biopsy, pancreatic biopsy, and spleen, bonemarrow or lymph node biopsy samples.

Further general aspects of the invention feature therapeutic methods andcompositions, including one or more modulators (stimulators or,preferably, inhibitors) of the expressed MRP-β gene and/or protein.Accordingly, the invention provides means for mitigating (detectablydecreasing or otherwise affecting) aberrant expression of an MRP-β gene,or aberrant production or biological function of an MRP-β polypeptide.The invention thus provides means for attenuating an undesirablephenotype, such as a disease-associated phenotype, that is contributedto by MRP-β. In preferred embodiments, the invention provides means forattenuating a multidrug-resistance phenotype, particularly a phenotypecontributed to by MRP-β. More particularly, a seventh aspect of theinvention features methods for mitigating aberrant expression of anMRP-β gene, and/or aberrant alteration or biological function of anMRP-β polypeptide. One embodiment involves the administration of anantisense pharmaceutical composition of the present invention to amammal suffering from effects of the aberrant phenotype associated withaltered expression and/or function of MRP-β. Another embodiment involvesthe administration of an antibody or fusion polypeptide of the presentinvention. In either embodiment, the therapeutic agent is administeredsystemically or locally under conditions sufficient to mitigate orattenuate the aberrant MRP-β associated phenotype. Preferably, thetherapeutic agent is administered under conditions sufficient to destroycells aberrantly producing MRP-β. In this manner, the invention providesmeans for destroying multidrug-resistant tumor cells in situ in the bodyof a mammal. In preferred embodiments, either of the foregoingtherapeutic agents can be administered as an adjuvant to conventionalchemotherapy. That is, either of the foregoing therapeutic agents can becoadministered together with one or more chemotherapeutic drugs. Thepresent antisense or fusion polypeptide therapeutic agent can beadministered prior to, concomitant with, or following administration ofone or more chemotherapeutic drugs. In such embodiments, the antisensepharmaceutical composition mitigates resistance of MRP-β expressingcells to the cytotoxic effects of the chemotherapeutic drug. That is,the antisense composition attenuates the MRP-β phenotype, which isexpected to be characterized by display of an ABC Transporter Proteinfamily member (MRP-β) and by the property of multidrug resistance. Thisis accomplished by disrupting activation or transcription of the MRP-βgene, or by destabilizing RNA transcripts thereof. Diminished ordiscontinued expression of MRP-β renders cells more susceptible to thecytotoxic effects of a chemotherapeutic drug that otherwise would beexported by MRP-β. Similarly, a therapeutically administered cytotoxicfusion polypeptide localizes in the vicinity of cells aberrantlydisplaying MRP-β, producing cytolysis thereof. A chemoattractant fusionpolypeptide also localizes to MRP-β displaying cells, stimulatingdestruction thereof by macrophages, killer T cells or cytotoxic T cells.

An eighth aspect of the invention features methods for identifying amodulator (a stimulator or, preferably, an inhibitor) of MRP-β. Thepresent modulator is useful for treating a disease or deleteriouscondition that is contributed to by MRP-β. Preferably, the modulator isa small molecule. In general, the present identification method relieson the use of an MRP-β expressing host cell produced as describedherein. Prokaryotic or eukaryotic host cells can be used for purposes ofidentifying an MRP-βmodulator, however in general, eukaryotic host cellsare preferred. Yeast or mammalian cells may be used, as desired or asdictated by specific circumstances. Presently, mammalian host cells,particularly human cells are preferred. The MRP-β expressing host cellis contacted with a candidate modulator, and after a sufficient periodof time for modulatory effects to be manifested, the cell is assayed todetermine whether the candidate indeed affects MRP-β. In one embodiment,the level of cellular MRP-β gene expression is assayed. A detectabledecrease (attenuation) or cessation (abrogation) in MRP-β geneexpression indicates that the candidate is an inhibitory modulator orinhibitor. Conversely, a detectable increase (augmentation) in MRP-βgene expression indicates that the candidate is a stimulatory modulatoror stimulator. Another embodiment involves assay of the amount or rateof production of MRP-β polypeptide displayed by the cell. A detectabledecrease or cessation of immunologically recognized MRP-β polypeptideindicates that the candidate is an inhibitory modulator. In a thirdembodiment, the host cell is contacted with a substrate (e.g., acytotoxin) exported or sequestered by MRP-β. The candidate inhibitor iscontacted with the host cell prior to, concomitantly with, or followingexposure to the substrate. The amount of substrate exported orsequestered by the cell is assessed. A detectable decrease in efflux orsequestration of the substrate indicates that the candidate is aninhibitory modulator. Alternatively, in specific embodiments wherein thesubstrate is cytotoxic, survival of the host cell is assessed. Adetectable decrease in survival indicates that the candidate is aninhibitory modulator. Candidate substances appropriate for screening asMRP-β modulators in any of the foregoing embodiments include natural orsynthetic metabolites, toxins, antibiotics, elements of a combinatorialchemistry, nucleotide or peptide library, naturally sourced cellsecretion products, cell lysates, and the like. Preferred substances forscreening, and preferred modulators, are small molecules.

Accordingly, a ninth aspect of the invention features an MRP-βmodulator, especially an inhibitory modulator, identified by any of theabove-described methods. Preferably, the modulator is a small molecule,e.g., an element of a combinatorial chemistry library or a low molecularweight natural or synthetic product or metabolite. The modulator may bedispersed in a pharmaceutically acceptable vehicle to produce amultidrug-resistance attenuating pharmaceutical composition of thepresent invention.

A tenth aspect of the invention thus features modulator-based methods ofmitigating aberrant MRP-β expression and/or polypeptide productionand/or biological function. The present method involves the step ofadministering an MRP-β modulator, optionally dispersed in apharmaceutically acceptable vehicle to a mammal suffering from effectsof the MRP-β associated aberrancy. Therapeutic modulation (preferablyinhibition) of MRP-β is useful for the treatment, including prophylaxis,remediation and palliation, of any disease or deleterious condition thatis contributed to by an abnormality affecting the MRP-β gene, itsexpression, MRP-β polypeptide production or biological function. In apreferred embodiment, the invention provides a method for improving(potentiating) effectiveness of chemotherapy to eradicate aberrantMRP-βexpressing cells, e.g., multidrug resistant transformed cells, fromthe body of a mammal. This method involves the steps of administering achemotherapeutic drug to the mammal, and coadministering an MRP-βmodulator identified as described herein. Preferably, the modulator isprovided in the form of a multidrug-resistance attenuating composition,i.e., dispersed in a pharmaceutically acceptable vehicle. This method isparticularly preferred where a chemotherapy adjuvant is desired toeradicate multidrug-resistant tumor cells. Advantageously, the methodcan be practiced where a fluid (e.g., leukemia, lymphoma, lymphsarcomaor ascites) tumor is present, or where the situs of a primary ormetastatic tumor is deemed unsuitable for surgical intervention orespecially where a remontant or reemergent tumor is observed followingan initial course of chemotherapeutic treatment. The present embodimentsare suitable for the treatment of any tumor, especially of mammary,respiratory tract, urogenital tract, endocrine system or immune systemorigin.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the presentinvention, as well as the invention itself, will be more fullyunderstood from the following description of preferred embodiments, whenread together with the accompanying drawings, in which:

FIG. 1A-G is a text representation of an MRP-β cDNA sequence and of thepolypeptide sequence encoded therein, as set forth in SEQ ID Nos: 1 and2.

FIG. 2A-F is a text representation comprising aligned amino acidsequences of the known ABC Transporter Protein superfamily member MRP(described in Deeley et al. (1996) U.S. Pat. No. 5,489,519), and of thenovel MRP-β disclosed herein. Dashes (-) indicate gaps introduced tomaximize alignment of similar sequences; colons (:) indicate thelocations of identical aligned amino acid residues.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Mammalian cells having a “multidrug-resistance” or “multidrug-resistant”phenotype are characterized by the ability to sequester, export or expela plurality of cytotoxic substances (e.g., chemotherapeutic drugs) fromthe intracellular milieu. Cells may acquire this phenotype as a resultof selection pressure imposed by exposure to a single chemotherapeuticdrug (the selection toxin). Alternatively, cells may exhibit thephenotype prior to toxin exposure, since the export of cytotoxicsubstances may involve a mechanism in common with normal export ofcellular secretion products, metabolites, and the like. Multidrugresistance differs from simple acquired resistance to the selectiontoxin in that the cell acquires competence to export additionalcytotoxins (other chemotherapeutic drugs) to which the cell was notpreviously exposed. For example, Mirski et al. (1987), 47 Cancer Res.2594-2598, describe the isolation of a multidrug-resistant cellpopulation by culturing the H69 cell line, derived from a human smallcell lung carcinoma, in the presence of adriamycin (doxorubicin) as aselection toxin. Surviving cells were found to resist the cytotoxiceffects of anthracycline analogs (e.g., daunomycin, epirubicin,menogaril and mitoxantrone), acivicin, etoposide, gramicidin D,colchicine and Vinca-derived alkaloids (vincristine and vinblastine) aswell as of adriamycin. Similar selection culturing techniques can beapplied to generate additional multidrug-resistant cell populations.

The functional property of multidrug-resistance is associated withexpression and cell-surface display of one or more ABC TransporterProtein superfamily members with energy-dependent export function (e.g.,P-glycoprotein, MRP or MRP-β as disclosed herein). The cell populationdescribed in Mirski et al. (1987) was reported in Cole et al. (1992),258 Science 1650-1654 to overexpress MRP (a correction of the reportedMRP sequence appears at 260 Science 879). Currently, antibodiesspecifically reactive with P-glycoprotein or MRP, or nucleic acid probesspecific for the corresponding expressed nucleic acid sequences, areused to ascertain the molecular basis of multidrug-resistance in a givencell population. Where the cell population in question includestransformed cells in the body of a cancer sufferer, determination of themolecular basis of the observed phenotype can assist the clinician inascertaining whether treatment with one of the so-called“chemosensitizers” or “MDR reversal agents,” the majority of whichaffect P-glycoprotein, is appropriate. Thus, knowledge of the molecularbasis of the observed phenotype provides information relevant todeveloping or revising a course of disease management. Zaman et al.(1993), 53 Cancer Res. 1747-1750, cautions, however, that the inductionor overexpression of MRP does not account for all forms ofmultidrug-resistance phenotype that are not attributable toP-glycoprotein expression. The discovery of MRP-β, reported herein,establishes that additional members of the ABC Transporter Proteinfamily exist in the mammalian (e.g., human) genome and likely contributeto the occurrence of multidrug-resistance in transformed cells.

MRP-β was identified by computer-assisted screening of a nucleic acidsequence database corresponding to a human endothelial cell cDNAlibrary. The library comprises cDNAs derived from RNA transcripts ofgenes expressed by differentiated endothelial cells cultured frommicrovascular tissue of mammary origin. The library was constructed, andnucleic acid components thereof were sequenced, by conventionaltechniques as set forth in Current Protocols in Molecular Cloning,Ausubel et al., eds. (1989), Greene Publishing and Wiley Interscience,New York, N.Y. The known sequence of MRP was used to query the databaseusing the TBLAST N algorithm disclosed in Altschul et al. (1990), 215 J.Mol. Biol. 403-410. The query sequence is disclosed in Cole et al.(1992), 258 Science 1650-1654 and 260 Science 879. See also SEQ ID No: 1of Deeley et al. (1996), U.S. Pat. No. 5,489,519, the disclosure ofwhich is incorporated by reference herein. The starting searchparameters for TBLAST N were as follows: score=200; word length=12.

The foregoing analysis identified a novel nucleic acid sequence withdetectable similarity to the query sequence. The novel sequence,disclosed herein as SEQ. ID No: 3, corresponds to a unique fragment of ahitherto unknown multidrug-resistance associated polypeptide, hereindesignated MRP-β. As defined herein, a “unique fragment” of a protein ornucleic acid is a peptide or oligonucleotide of sufficient length tohave a sequence unique to a particular gene or polypeptide, i.e., asequence not shared by related or unrelated genes or polypeptides. Thus,for example, a unique nucleic acid fragment typically will have at least16 nucleotide residues, and a unique polypeptide fragment typically willhave at least 6 amino acid residues. Preferably, to ensure substantiallyunique occurrence in a typical higher eukaryotic genome, a uniquenucleic acid fragment should have at least 20 nucleotide residues, and aunique polypeptide fragment should have at least 8 amino acid residues.Unique polypeptide fragments are referred to herein as epitopes. The SEQID No: 3 unique fragment of MRP-β nucleic acid is 465 nucleotideresidues in length and has a sequence approximately 62% identical tothat of the corresponding aligned fragment of the MRP gene. In contrast,SEQ ID No: 3 lacks detectable similarity to the product of the MDR1gene, P-glycoprotein.

A nucleic acid probe was prepared using the SEQ ID No: 3 sequence, asdescribed in EXAMPLE 1 herein, and used for hybridization screening ofan appropriate expression (cDNA) library. The screen yielded an MRP-βcDNA having the sequence set forth as nucleotides 674847 of SEQ ID No: 1herein. This cloned cDNA has been designated fohd013a05m and has beendeposited (Apr. 16, 1997) in the American Type Culture Collection underthe terms of the Budapest Treaty. The sequence of fohd013a05maccordingly is incorporated herein by reference. The original SEQ ID No:3 fragment corresponds generally to nucleotides 3701 to 4144 of thecloned SEQ ID No: 1 cDNA. The full-length MRP-β cDNA extends a shortdistance upstream (5′) of the fohd013a05m MRP-β insert. The MRP-βtranscript produced in human cells and/or tissue is approximately 6 kb,as visualized in the Northern blot studies described in EXAMPLES 2 and3. A cDNA comprising 66 nucleotides upstream (5′) of the fohd013a05mMRP-β insert was isolated as described in EXAMPLE 1. The cDNA sequencepresented in SEQ ID No: 1 comprises the sequence of the fohd013a05mMRP-β insert and the 66 upstream nucleotides. The native 5′ end of thecellular MRP-β transcript can also be elucidated readily using a 5′-RACEprotocol known in the art, for example as described in Siebert et al.(1995), 23 Nucl. Acids Res 1087-1088, and in the Clonetech, Inc. UserManual for Marathon-Ready cDNA (1996), the teachings of which areincorporated herein by reference.

The present invention encompasses all MRP-β nucleic acids that can beisolated or constructed by conventional molecular engineeringtechniques, using the information made available as a result of thepresent disclosure. Thus, for example, the invention encompasses nucleicacids comprising sequences complementary to all or a unique fragment ofthe SEQ ID No: 1 cDNA. The sequence of a “complementary” nucleic acidstrand is composed of the Watson-Crick base pair partners of thenucleotide residues in a specified nucleic acid, i.e., a guanidine (G)residue corresponding to each cytosine (C) residue in the specifiednucleic acid, and an adenine (A) residue corresponding to each thymidine(T) or uracil (U) residue therein. Thus, the invention encompasses RNAhaving a sequence complementary to SEQ ID No: 1. The present RNA can beobtained as a cell-free lysate or extract (e.g., as described in EXAMPLE2), or can be isolated in substantially pure form using techniquesdescribed in Current Protocols in Molecular Cloning, Ausubel et al.,eds. (1989), Greene Publishing and Wiley Interscience, New York, N.Y.and in Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.The invention further encompasses a nucleic acid probe or primer havinga nucleotide sequence complementary to a unique fragment of the MRP-βgene described herein. The probe optionally further comprises adetectable moiety, or creates a detectable complex, when hybridized tothe target (MRP-β) sequence. Non-limiting examples of appropriatedetectable moieties including fluorophores (e.g., fluorescein,rhodamine, Texas Red and the like), radionucleotides (e.g., ³H, ¹⁴C, ³²Pand the like), and binding-pair partners (e.g., biotin, avidin orstreptavidin). The probe or primer need not be strictly complementary tothe target sequence: it is only necessary that a sufficient number ofprobe nucleotides be capable of forming base pairs with targetnucleotides to produce a stable, double-stranded nucleic acid complexunder hybridization conditions.

Hybridization is the noncovalent, antiparallel bonding of complementarynucleic acid strands, in which Watson-Crick base pairing is established.To ensure specificity, hybridization should be carried out understringent conditions, defined herein as conditions of time, temperature,probe length, probe and/or target concentration, osmotic strength, pH,detergent, carrier nucleic acid, etc. that permit no more than anoccasional base-pairing mismatch within a probe/target duplex. Highlystringent conditions exclude all but about one base pair mismatch per kbof target sequence. Exemplary highly stringent conditions involvehybridization to membrane immobilized target nucleic acid at atemperature of 65° C. in the presence of 0.5 m NaHPO₄, 7% SDS, 1 mMEDTA, followed by washing at 68° C. in the presence of 0.1×SSC, 0.1%SDS. Current Protocols in Molecular Biology (1989), Ausubel et al.,eds., Greene Publishing and Wiley Interscience, New York, N.Y. Incircumstances where relatively infrequent mismatches, e.g., up to aboutten mismatches per kb of target, can be tolerated, moderately stringentconditions may be used. For moderate stringency, probe/target hybridsformed under the above conditions are washed at 42° C. in the presenceof 0.2×SSC, 0.1% SDS. The invention encompasses all nucleic acids thathybridize to nucleic acid, the sequence of which comprises SEQ ID No: 1or a unique fragment thereof.

Nucleic acids that are complementary to or hybridize to all or a uniquefragment of the novel MRP-β gene can be used as antisense or primeroligonucleotides. Antisense oligonucleotides disrupt gene expressionand/or protein production and thereby attenuate an aberrant phenotypeattributable to inappropriate expression or activation of the targetgene. Lautier et al. (1996), 52 Biochem. Pharmacol. 967-977. As aresult, the phenotype is abrogated or its penetrance is diminished(attenuated). Therapeutic intervention to attenuate amultidrug-resistance phenotype, for example, restores cellularvulnerability to cytotoxic drugs. Smyth et al. (1996), PCT Publ. WO96/02556, teaches that antisense oligonucleotides disrupt expression ofthe target gene by interfering with gene transcription, transcriptsplicing, or translation; by triggering enzymatic destruction by RNAseH; or by destroying the target through one or more reactive moietiesincorporated into the antisense compound. Preferred oligonucleotidesherein have sequences sufficiently complementary to all or a uniquefragment of the MRP-β gene to hybridize, under intracellular conditions,to the gene's coding or noncoding strand, or to an RNA transcript of thegene. Optionally, the oligonucleotide can be designed to hybridize to apolypeptide coding region, or to a 5′ or 3′ untranslated region of thegene or gene transcript, or to a gene intron or an intron/exon boundary.Typically, the present oligonucleotides are at least 9 nucleotides inlength, and range from about 12 to about 40 bases in length, and aregenerally about 16 to 30 bases in length, with about 20 bases beingconsidered optimal. Exemplary oligonucleotides are at least 15, 21, or24 nucleotides in length. Specific examples of the presentoligonucleotides are set forth in SEQ ID Nos: 4, 5, 6, 7 and 8. Theseand other exemplary oligonucleotides can be synthesized readily byconventional techniques.

While either DNA or RNA is suitable for use in primer, probe orantisense oligonucleotides, it is often desirable to include one or moremodified bases, backbone sugar moieties, or backbone linking groups.Thus, Smyth et al. (1996) teaches that alkylphosphonates,phosphorothioates, phosphorodithioates, phosphate esters,alkylphosphonothioates, phosphoramidates, carbamates, carbonates,phosphate triesters, acetamidate, 2-O-methyls, and carboxymethyl estersall are suitable for use in the context of antisense oligonucleotides.Preferred modified olignoculeotides herein comprise a modified backbonestructure. Peptide nucleic acid (PNA) oligonucleotides preparedaccording to the teachings of Perry-O'Keefe et al. (I 996), 93 Proc.Nat'l. Acad. Sci. USA 14670-14675, and Egholm et al. (1993), 365 Nature566-568, are particularly preferred herein.

In addition, the invention encompasses all MRP-β nucleic acids havingsequences at least 50% identical to SEQ ID No: 1 or to the complementthereof. The determination of whether a particular sequence meets thiscriterion is made using the TBLAST N algorithm according to theteachings of Altschul et al. (1990), 215 J. Mol. 403-410, the teachingsof which are incorporated herein by reference. Such nucleic acids encodevariants, which may be nat lly-occurring or biosynthetic, of theMRP-βpolypeptide disclosed herein.

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino acid ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”). The percenthomology between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100). The determination ofpercent homology between two sequences can be accomplished using amathematical algorithim. A preferred, non-limiting example of amathematical algorithim utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12 to obtain nucleotide sequences homologous to D6nucleic acid molecules of the invention. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to D6 protein molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See, for example, the National Centersfor Biotechnology Information website which is linked to the NationalLibrary of Medicine website (www.ncbi.nlm.rih.gov). Another preferred,non-limiting example of a mathematical algorithim utilized for thecomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM 120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Similarly, the invention encompasses all nucleic acids which, by virtueof the well-known degeneracy of the genetic code, also encode the SEQ IDNo: 2 polypeptide. Such degenerate variants may be naturally-occurringor may be produced through routine application of molecular engineeringtechniques. Current Protocols in Molecular Cloning, Ausubel et al., eds.(1989), Greene Publishing and Wiley Interscience, New York, N.Y. and inSambrook et al. (1989), Molecular Cloning A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Furthermore, the invention encompasses all nucleic acids encodingpolypeptides having sequences that share at least 75% sequencesimilarity with the disclosed MRP-β polypeptide. Similarity iscalculated generally according to the method of Altschul et al. (1990),215 J. Mol, Biol, 403-410, using the TBLAST P algorithm. Moderate gapsor insertions of amino acid residues are ignored for similaritycalculation purposes. Preferably, the MRP-β variants encoded by thesenucleic acids function similarly to MRP-β when expressed by a host cellproduced as described herein. That is, preferred MRP-β variantpolypeptides are displayed on the surface of a host cell and contributeto the cell's acquisition of a multidrug-resistance phenotype. MRP-βvariants thus may differ from that comprising SEQ ID No: 2 by thepresence of one or more amino acid insertions, deletions, or pointsubstitutions. Deletion variants are expected to facilitateinvestigation into the minimum MRP-β polypeptide structure required tosupport drug transport and thus multidrug-resistance phenotype.Substitution variants are expected to facilitate investigation into themechanism and specificity of MRP-β function. Exemplary substitutionvariants include chimeric polypeptides in which one or more MRP-β aminoacid residues are replaced by the corresponding residue in either theMRP or P-glycoprotein sequence. All nucleic acids encoding such variantsare within the scope of the present invention. All oligonucleotidescomplementary to, or which hybridize to, the present nucleic acids arewithin the scope of this invention.

All of the foregoing nucleic acids of the present invention can beproduced, expressed, and/or manipulated by conventional molecularengineering techniques such as the techniques set forth in CurrentProtocols in Molecular Cloning, Ausubel et al., eds. (1989), GreenePublishing and Wiley Interscience, New York, N.Y. and in Sambrook et al.(1989), Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., and the teachingsdescribed and referenced in Watson et al. (1992), Recombinant DNA, 2nded., Scientific American Books and W. H. Freeman & Co., New York, N.Y.

Any of the foregoing nucleic acids can be inserted into an expressionvector by routine molecular engineering techniques. Current Protocols inMolecular Cloning, Ausubel et al., eds. (1989), Greene Publishing andWiley Interscience, New York, N.Y. and in Sambrook et al. (1989),Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., and publications referencedin Watson et al. (1992), Recombinant DNA, 2nd ed., Scientific AmericanBooks and W. H. Freeman & Co., New York, N.Y. Preferred expressionvectors thus encode full-length or unique fragment MRP-β polypeptides.Particularly preferred are expression vectors that, when expressed in asuitable host cell, contribute to the emergence of amultidrug-resistance phenotype therein. In other embodiments, the vectorcomprises DNA or RNA complementary to an antisense oligonucleotide. Thepresent expression vectors further comprise one or more conventionalexpression control elements, such as an enhancer, promoter, initiationsite, or termination site operatively associated with the inserted MRP-βnucleic acid. Non-limiting examples of suitable expression controlelements include the cytomegalovirus immediate early gene, the early orlate promoters of SV40 adenovirus, the lac system, the trp system, theTAC system, the TRC system, the major operator and promoter regions ofphage A, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase, the promoter of acid phosphatase, thepromoters of yeast α-mating factors, and immunoglobulin enhancers and/orpromoters. Optionally, the expression vector may comprise a selectionmarker, such as an antibiotic resistance gene. Single or multiple copiesof the inserted MRP-β nucleic acid can be encoded by the vector.Preferably, for production of eukaryotic (preferably mammalian) hostcells, or for therapeutic purposes, the vector is retroviral in originor comprises one or more retroviral elements. The vector can be taken up(internalized) by cells via transfection, infection, microinjection,pinocytosis or in the course of cell division, or can be packaged, e.g.,in a liposome or retroviral envelope. In this manner, the vector can bedesigned for selective internalization in dividing cells, transformedcells, or in cells of a tissue type susceptible to retroviral infection.Deeley et al. (1996), U.S. Pat. No. 5,489,519, the teachings of whichare incorporated herein by reference, summarizes conventional techniquesfor the preparation of expression vectors.

The present MRP-β expression vectors are suitable for use in anyconventional host cell transfection technique, e.g., as described inCurrent Protocols in Molecular Cloning, Ausubel et al., eds. (1989),Greene Publishing and Wiley Interscience, New York, N.Y. and in Sambrooket al. (1989), Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and inpublications referenced in Watson et al. (1992), Recombinant DNA, 2nded., Scientific American Books and W. H. Freeman & Co., New York, N.Y.Thus, the present invention further provides a host cell that producesan MRP-β polypeptide or an MRP-βantisense oligonucleotide. Preferredhost cells display an MRP-β polypeptide on the cell surface and/ordisplay a multidrug-resistance phenotype. Such host cells are expectedto facilitate elucidation of the types or structural classes ofchemotherapeutic drugs or other substances ejected or sequestered fromthe intracellular milieu by MRP-β. Thus, MRP-β host cells allow rapid,in vitro evaluation of the specific characteristics of themultidrug-resistance phenotype associated with MRP-β expression oroverexpression. Such cells further allow production of MRP-βpolypeptides and antibodies as described below.

Cells (source cells) suitable for the production of the foregoing hostcells include, but are not limited to, primary or immortalizedepithelial cells such as carcinoma cells or cell lines. Additionalsource cells include primary or immortalized mesenchymal cells, such assarcoma cells. Still further suitable cells include hematopoietic systemcells, such as leukemia, lymphoma or lymphosarcoma cells. Mammalian ornon-mammalian cells can be used, but in general, mammalian (e.g.,murine, ovine, porcine, bovine or, preferably, human) cells arepreferred. For certain purposes, such as the rapid phenotypiccharacterization of deleterious phenotypes (e.g., multidrug resistancephenotypes) conferred by MRP-β alteration, expression or overexpression,or such as the rapid screening of candidate modulators of MRP-β,non-mammalian cells such as insect cells or yeast cells, also may beused. In all circumstances, the identification of transfectants (newlyproduced host cells) is dependent on the use of source cells that arevulnerable to the cytotoxic effects of drugs transported by MRP-A ormetabolized by the product of a selection marker gene optionallyincluded in the vector.

Deeley et al. (1996), 5,489,519, Cole et al. (1994), 54 Cancer Res.5902-5910, and Stride et al. (1995), 49 Mol. Pharmacol. 962-971, eachdescribe the transfection of human HeLa cells with MRP to produce an MRPexpressing host cell. Engel et al. (1996), U.S. Pat. No. 5,556,856, andZelle et al. (1996), U.S. Pat. No. 5,543,423, describe the transfectionof murine leukemia cells with MDR-1 to produce P-glycoprotein expressinghost cells. Sarkadi et al. (1995), PCT Publ. WO9531474 describes thetransfection of murine NIH 3T3 fibroblasts and of Spodoptera frugiperda(insect) cells with MDR-1 to produce P-glycoprotein expressing hostmurine and insect cells, respectively. Ruetz et al. (1996), 271 J. Biol,Chem, 4154-4160, describes the transfection of Saccharomyces cerevisiae(yeast) with MRP and MDR-1 to produce yeast host cells. Any of theabove-mentioned, available source cells can be transfected according tostandard techniques with an MRP-β expression vector to produceMRP-βexpressing host cells. Relevant techniques are disclosed in theabove-cited references and in Current Protocols in Molecular Cloning,Ausubel et al., eds. (1989), Greene Publishing and Wiley Interscience,New York, N.Y. Currently, the immortalized MCF-7 human breastadenocarcinoma cell line, available from the American Type CultureCollection as ATCC No. HTB22, is a preferred source cell. An exemplarystandard transfection technique suitable for use with MCF-7 is thelipofectin technique summarized in Cole et al. (1994), however, manyconventional alternatives (e.g., calcium phosphate; lithium acetate;baculoviral or retroviral infection) are available and can be used withthe MCF-7 or other exemplary source cell lines. After transfection,transfectants can be identified by culturing the cells in the presenceof hygromycin B (as in Cole et al. (1994)) or another selection toxin,such as bisantrene or adriamycin (doxorubicin). Expression of abiologically-functional MRP-β polypeptide can be confirmed by analyzingcellular RNA for the presence of vector-derived MRP-β transcripts; byanalyzing cellular protein for the presence of an epitope unique to,MRP-β; by analyzing the cell surface for display of an epitope unique toMRP-β; or, by analyzing whether the cell has acquired an MRP-βassociated phenotype, such as a multidrug-resistance phenotype.

The present host cells initially are expected to facilitate productionof MRP-β polypeptides and structural and functional analysis thereof.The MRP-β polypeptide comprising SEQ ID No: 2 is expected to bind ATP,and to be an integral, multispanning transmembrane protein generally asdescribed in Almquist et al. (1995), 55 Cancer Res. 102-110. Asignificant portion of the total MRP-β produced in host cells isexpected to span the cells' plasma membrane, with an additional portionbeing present intracellularly, e.g., in the endoplasmic reticulum and/orthe Golgi apparatus. Thus, MRP-β host cells are expected to displayextracellular portions of the multispanning MRP-β polypeptide on thecell surface, appropriately configured to mediate the ATP-dependentsequestration or export (efflux) of a plurality of cytotoxic drugs,including drugs conventionally used as chemotherapeutic agents. Thesegeneral properties are deduced from an assessment of the primarystructure (sequence) of the MRP-β polypeptide. MRP-β is considered to bea novel member of the ABC Transporter Protein superfamily and is deemedlikely to contribute to multidrug-resistance phenotypes by mediatingdrug transport across cellular phospholipid membranes. FIG. 2A-F setsforth an exemplary sequence alignment of the disclosed novel MRP-βpolypeptide (SEQ ID No: 2), with relevant sequence of the MRPpolypeptide of Deeley et al. (1996), U.S. Pat. No. 5,489,519 (SwissProtP33527, 1531 aa). The alignment was generated using the ALIGN algorithm(which calculates a global alignment of two sequences), version 2.0(Myers and Miller (1989) CABIOS), scoring matrix: PAM120, gap penalties:−12/−4, 30.9% identity, global alignment score: 1214.

The present host cells provide an appropriate purification source forobtaining useful quantities of MRP-β polypeptide. The polypeptide can beisolated in substantially pure form (i.e., essentially free ofdetectable levels of non-MR-β polypeptides or other cell components) byan appropriate combination of one or more protein extraction orpurification techniques such as those described in Sambrook et al.(1989), Molecular Cloning A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. Alternatively, anMRP-β enriched subcellular membrane preparation can be obtained bysuitable cell disruption and fractionation techniques, all of which arewell-known in the art. An exemplary, adaptable protocol for obtaining anMRP enriched subcellular membrane preparation is set forth in Zaman etal. (1994), 91 Proc. Natl. Acad. Sci. USA 8822-8826. Intact host cells,MRP-β enriched membrane preparations thereof, and/or isolated MRP-βprotein can be used for a number of purposes, such as the production ofmonoclonal or polyclonal antibodies, characterization of substratesaffected by MRP-β biological function, and identification of novelmodulators (e.g., inhibitors) affecting MRP-β biological function.

Antibody production involves administration of one or more immunogenicdoses of an MRP-β polypeptide preparation (whether isolated orincorporated in a cell membrane) to an appropriate non-human animal,such as a mouse, rat, rabbit, guinea pig, turkey, goat, sheep, pig, orhorse. To enhance immunogenicity, the preparation can be emulsified witha conventional adjuvant, such as Freund's complete or incompleteadjuvant. Routine monitoring of serum immunoglobulins, using peripheralblood samples withdrawn at appropriate intervals (e.g., seven to tendays) after an initial or subsequent immunization, can be used to detectthe onset and/or maturation of a humoral immune response. Detection and,optionally, quantitation, of immunoglobulins selectively reactive withan MRP-β epitope can be achieved through any conventional technique,such as ELISA, radioimmunoassay, Western blotting, or the like.Appropriate means of eliciting and monitoring production of antibodieswith selective reactivity (binding) for other multidrug-resistanceassociated proteins are disclosed in Arceci et al. (1994), U.S. Pat. No.5,369,009, which is incorporated herein by reference. An immunoglobulin“selectively reactive with an MRP-β epitope” has, binding specificityfor the recognized epitope such that an antibody/epitope complex formsunder conditions generally permissive of the formation of such complexes(e.g., under conditions of time, temperature, ionic strength, pH, ionicor nonionic detergent, carrier protein, etc.). Serial dilution(titration) analysis by standard techniques is useful to estimate theavidity of antibodies in the immune serum sample for one or moreepitopes unique to MRP-β. As defined herein, an “epitope unique toMRP-β” is a unique, immunogenic fragment of the full-length MRP-βpolypeptide. A unique linear epitope typically ranges in size from aboutten to about twenty-five amino acid residues, and frequently is abouttwelve to eighteen residues in length. Unique conformational epitopesalso are provided herein, and comprise two or more unique fragments ofthe MRP-β polypeptide that, due to their juxtaposition in the foldedpolypeptide, form a single immunogenic epitope.

Immune serum having a high titer generally is preferred herein. Serumhaving a half-maximal avidity for a unique MRP-β epitope of at leastabout 1:1000, preferably at least about 1:110,000, can be harvested inbulk for use as a source of polyclonal antibody useful in the detectionand/or quantitation of MRP-β. Polyclonal immunoglobulins can, ifdesired, be enriched by conventional fractionation of such serum, or canbe isolated by conventional immunoadsorbent techniques, e.g., using aProtein A or Protein G chromatography resin. Immune, high titer murineor guinea pig serum alternatively is preferred herein for the productionand screening of hybridomas secreting monoclonal antibodies selectivelyreactive with MRP-β. The present hybridomas can be produced according towell-known, standard techniques. The present monoclonal antibodies canbe obtained from hybridoma culture supernatant, or from conventionallyproduced ascites fluid, and optionally isolated via immunoadsorbentchromatography or another suitable separation technique prior to use asagents to detect and/or quantitate MRP-β.

A preferred antibody, whether polyclonal or monoclonal, is selectivelyreactive with a unique MRP-β epitope that is displayed on the surface ofMRP-β expressing cells, such as a host cell as provided herein. Thepreferred antibody accordingly can be used to detect and, if desired,quantitate MRP-β expressing cells, e.g., normal or transformed cells ina mammalian body tissue or biopsy sample thereof. Exemplary analogousmethods for the use of antibodies reactive with epitopes unique toP-glycoprotein are disclosed in Arceci et al. (1994), U.S. Pat. No.5,369,009; exemplary analogous methods for the use of antibodiesreactive with epitopes unique to MRP are disclosed in Deeley et al.(1996), U.S. Pat. No. 5,489,519. Both disclosures are incorporatedherein by reference. Specifically, the preferred antibody can be used todetect MRP-β expressing cells whether such cells are host cells ormammalian body tissue cells that aberrantly express MRP-β as a result ofexposure to a selection toxin such as a chemotherapeutic drug.Advantageously, intact, e.g., living, cells that display a unique MRP-βepitope can be detected by standard immunohistochemical, radiometricimaging or flow cytometry techniques. The present antibody can be usedto detect and/or monitor MRP-β polypeptide production in lieu of or inaddition to detecting MRP-β gene expression using the novel MRP-βnucleic acids provided herein. Thus, the antibody can be used to assesswhether an aberrant phenotype, such as a multidrug-resistance phenotype,in a given cell population is associated with cell surface display ofMRP-β. Further, the antibody can be used to assess the naturaltissue-specific production of MRP-β, and thus to assess tissues likelyto give rise to multidrug-resistant carcinomas or sarcomas. In addition,the present antibody can be used to monitor tumor biopsy samples toprovide information relevant to selecting or revising a course ofdisease management, or to diagnosis, prognostication and/or staging ofany disease associated with an abnormality affecting MRP-β. An exemplarydisease is proliferative neoplastic disease. Furthermore, the presentantibody can be used in a cell-sorting procedure or other cell isolationprocedure to generate a substantially pure preparation of MRP-βexpressing cells, or a cell population substantially depleted of MRP-βexpressing cells. Each of the foregoing can be achieved through routinepractice or modification of well-known techniques, including but notlimited to the conjugation of a detectable moiety (e.g., a radionuclide,fluorophore, chromophore, binding pair member, or enzyme) to the MRP-βreactive antibody.

A hybridoma secreting an MRP-β reactive monoclonal antibody of thepresent invention additionally provides a suitable source of nucleicacid for the routine construction of a fusion polypeptide comprising anantigen-binding fragment derived from the MRP-β reactive antibody. Thepresent fusion polypeptide can be prepared by routine adaptation ofconventional techniques therefor in Deeley et al. (1996), U.S. Pat. No.5,489,519 (incorporated herein by reference). The fusion polypeptide canbe a truncated immunoglobulin, an immunoglobulin having a desiredconstant region (e.g., IgG in lieu of IgM), or a “humanized”immunoglobulin having an MRP-β reactive Fv region fused to a frameworkregion of human origin. Additional fusion polypeptides can comprise, inaddition to an MRP-β reactive antigen-binding fragment, anon-immunoglobulin polypeptide such as a cytotoxic polypeptide (e.g.,diphtheria toxin, ricin) or a chemoattractant polypeptide thatstimulates immune effector cells (cytotoxic T cells, natural killercells, macrophages) to kill cells that display MRP-β. Standardtechniques well-known in the art can be used to produce appropriateimmunoglobulin fusion polypeptides of the present invention.

The foregoing compositions can be used for a number of purposes,including the assessment (e.g., for diagnostic purposes) ofabnormalities in the structure and/or expression of a cellular MRP-βgene. Thus, for example, the invention provides a method for detectingan abnormality in a cellular MRP-β gene, such as a mutation arising ingermline or somatic cellular genomic DNA. Similarly, the present methodprovides a means for detecting chromosomal rearrangement, restrictionfragment polymorphism, allelic loss or disruption of a nativemethylation pattern in the MRP-β gene. This method exploits thehybridization properties of an oligonucleotide probe or primer describedherein. A preferred oligonucleotide is modified by the presence of adetectable label and/or a peptide nucleic acid backbone. Sucholigonucleotides, which hybridize to one or more unique fragments of acellular MRP-β gene suspected of harboring a structural (e.g., sequence)abnormality, can be used in a diagnostic protocol as disclosed inPerry-O'Keefe et al. (1996); 93 Proc. Nat'l. Acad. Sci. USA 14670-14675,or as disclosed in Ravnik-Glavac et al. (1994), 3 Hum. Mol. Biol. 801-—.Other nucleic acid-based diagnostic methods that can be exploited forpurposes of assessing MRP-β gene abnormalities are as set forth in Myerset al. (1985), 230 Science 1242; Cotton et al. (1988), 85 Proc, Nat'l.Acad. Sci. USA 4397; Suleeba et al. (1992), 217 Meth. Enzymol 286-295;Orita et al. (1989), 86 Proc, Nat'l. Acad. Sci. USA 2766; Cotton et al.(1993), 285 Mutat. Res. 125-144; Hayashi (1992), 9 Genet. Anal. Tech.Appl. 73-79; and, Myers et al. (1985), 313 Nature 495. Additionalmethods are based on selective amplification and/or extension of MRP-βPCR primers, e.g., as described in Landegran et al. (1988), 241 Scienc1077-1080; Nakazawa et al. (1994), 91 Proc. Nat'l. Acad. Sci. USA360-364; and Abravaya et al. 91995), 23 Nucl. Acids Res. 675-682, and inpublications referenced in Watson et al. (1992), Recombinant DNA 2nded., Scientific American Books and W.H. Freeman & Co., New York, N.Y.

Additional diagnostic and/or characterization methods using nucleic acidcompositions provided herein include Northern blot, slot blot or similarmethods for visualizing fluctuations, especially abnormaloverproduction, in the level of cellular transcripts comprising MRP-βsequences. These methods rely on the use of MRP-β oligonucleotide probesand hybridization conditions appropriate for the formation of probe/RNAhybrids. Exemplary conditions for use with nucleic acid or modifiednucleic acid probes are as set forth in Perry-O'Keefe et al. (1996), 93Proc. Nat'l. Acad. Sci. USA 14670-14675; Current Protocols in MolecularCloning, Ausubel et al., eds. (1989), Greene Publishing and WileyInterscience, New York, N.Y. and Sambrook et al. (1989), MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. An exemplary transcript hybridizationprotocol is set forth in EXAMPLE 2 herein. This example confirms theassociation of MRP-0 expression with the occurrence of amultidrug-resistance phenotype transformed cell populations. Similarconfirmation can be obtained by comparing a normal cell population witha tissue-matched transformed multidrug resistant population. Preferably,the cell populations each are derived from an exemplary mammalian bodytissue, such as an epithelial tissue (e.g., mammary, respiratory tract,gastrointestinal tract, urogenital tract, paracrine, endocrine orneuroendocrine tissue). EXAMPLE 2 demonstrates that MRP-β expression issignificantly elevated in multidrug-resistant derivatives of well-knowncell fines, including the MCF-7 breast adenocarcinoma cell line, theHL60 promyelocytic leukemia cell line, the A2780 ovarian carcinoma cellline, and the U937 myeloid leukemia cell line. Thus, MRP-β expressionlevel correlates with the occurrence of multidrug-resistance rather thanwith derivation from a particular body tissue type.

Cellular MRP-β gene expression level similarly is expected to correlatewith the maintenance or reappearance of multidrug resistance intransformed cells in situ following exposure to one or morechemotherapeutic drugs, or to a conventional chemosensitizer or “MDRreversal” agent. In other words, MRF-β gene expression activation ortranscript stabilization is deemed likely to provide transformed cellswith a selective advantage that is distinct from the advantage(s)derivable from P-glycoprotein or MRP expression. As a result, themonitoring of MRP-β transcript or polypeptide production, or geneexpression level, or fluctuations therein, in one or more tumor biopsysamples is expected to provide information relevant to diagnosis,prognostication and/or staging of neoplastic disease in a cancersufferer. Any suitable means for detecting MRP-β transcript orpolypeptide production or stabilization, or gene expression level, canbe applied for the present diagnostic purposes. Thus, gene expressioncan be monitored using any appropriate nucleic acid based methoddescribed above. MRP-β polypeptide production or accumulation can bemonitored using an MRP-β antibody described herein. Any appropriateconventional method for visualizing selective binding of an antibody toits cognate epitope may be used. Appropriate methods are described inSambrook et al. (1989), Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

In some embodiments, diagnosis is achieved by hybridization techniquesinvolving the use of a modified MRP-β probe as described herein. Apreferred technique involved the use of a peptide-nucleic acid probe asdescribed in Egholm et al. (1993), 365 Nature 566-568, and Perry-O'Keefeet al. (1996), 93 Proc. Natl. Acad. Sci. USA 14670-14675. Thus, forexample, the protocol of EXAMPLE 2 can be routinely adapted to allowassessment of multidrug-resistant transformed cells that have survivedexposure in situ to a chemosensitizer or to an agent that interfereswith P-glycoprotein or MRP expression. Further exemplary demonstrationscan be produced by routinely adapting the EXAMPLE 2 protocol to theassessment of two or more biopsy samples obtained from an individual(e.g., a cancer sufferer) at different times. Preferably, a first biopsysample corresponds to a time of diagnosis or to a time prior to orconcomitant with the onset of chemotherapy. A second biopsy samplecorresponds to a timepoint at which beneficial results of chemotherapyare expected to be detectable (e.g., a time sufficiently following theonset of chemotherapy for cytotoxic effects to be observed). One or moresubsequent biopsy samples may correspond to further timepointsoptionally correlated with fluctuations in clinical parameters (e.g.,relapse, remission, a change in disease staging, or the like). Changes(fluctuations) in MRP-β gene expression, transcript stabilization,polypeptide production, and/or polypeptide stabilization are expected tocorrelate with, or to predict, the emergence or attenuation of adeleterious phenotype associated with MRP-β, such as amultidrug-resistance phenotype.

It will be appreciated that the causes of multidrug-resistancephenotypes vary with each individual cell type and are not whollyaccounted for by expression or overexpression of P-glycoprotein, MRP orthe novel MRP-β disclosed herein. Rather, additional members of the ABCTransporter Protein family may be involved, as may be one or moremembers of known or novel signal tranduction pathways or intracellularmetabolic or growth-regulatory pathways. The present discovery of MRP-βfacilitates investigation into the role(s) of such additional geneexpression products in the acquisition and/or maintenance of amultidrug-resistance phenotype. Specifically, the discovery of MRP-βprovides an improved method of identifying a gene, especially a hithertounknown gene, expression of which contributes to emergence ormaintenance of drug-resistant phenotype in transformed mammalian cells.

The identification method is an adaptation of the differential displaytechnique disclosed in Liang et al. (1997), U.S. Pat. No. 5,599,672 andPardee et al. (1993), U.S. Pat. No. 5,262,311. The method involves thesteps of providing a transformed or normal cell population (the firstpopulation) derived from an exemplary mammalian tissue, such as asecretory epithelium (a nonlimiting example of which would be mammaryepithelium), and culturing the cell population in the presence of aselection toxin, such that a drug-resistant derivative population (thesecond population) is produced. Mirsky et al. (1987), 47 Cancer Res.2594-2598, provides an exemplary protocol for selecting a drug-resistantderivative of an immortalized human small cell lung carcinoma cell line,H69. This exemplary protocol can be adapted to use with additional celllines, or with primary cells in culture. Thus, Hait et al. (1992), U.S.Pat. No. 5,104,858, teaches the stepwise selection of adoxorubicin-resistant derivative of the well-known MCF-7 breastadenocarcinoma cell line. Further adaptations include, e.g., the use ofa selection toxin other than adriamycin. Powell et al. (1995 and 1996),U.S. Pat. Nos. 5,387,685, 5,550,149 and 5,561,141, teaches the use ofbisantrene to select for a multidrug-resistant derivative of the knownhuman ovarian carcinoma cell line, OVCAR-3 (HTB-161). If desired, thefirst and second populations can be selected from well-known cell linesand/or available multidrug-resistant derivatives thereof. Sunkara(1996), U.S. Pat. No. 5,523,304, teaches the use of amultidrug-resistant human epidermoid carcinoma cell line, KBV1. Ramu etal. (1993), U.S. Pat. No. 5,190,946, teaches the use of a murineleukemia cell line (P388) and an available multidrug-resistantderivative thereof (P388/ADR). Alternatively, the populations can beselected from biopsy samples withdrawn from an individual (e.g., acancer sufferer) before and after a clinical observation of multidrugresistance. Currently, the MCF-7 cell line and multidrug-resistantderivatives thereof are considered exemplary and are preferred foranalysis of multidrug-resistance phenotypes.

Expressed nucleic acids (transcription products; RNA) are isolatedseparately from the first and second populations, and fractionated byelectrophoretic resolution or another conventional technique asdescribed in Liang et al. (1997), U.S. Pat. No. 5,599,672.Alternatively, the expression products of the first population are usedas an adsorbent to deplete the expression products of the secondpopulation of individual transcripts that are common to bothpopulations. Thereafter, the resolved expression products are analyzedto identify one or more gene transcripts that are preferentiallyexpressed, underexpressed or overexpressed in the second population.Such gene transcripts accordingly are associated with themultidrug-resistance phenotype. One or more probes complementary to thenovel MRP-β nucleic acids disclosed herein thus can be used as aninternal control to monitor successful identification ofmultidrug-resistance associated gene transcripts. Of course, probescomplementary to nucleic acids encoding P-glycoprotein and/or MRP can beused similarly. Multidrug-resistance associated gene transcripts thatare identified in this adaptation of the Liang et al. (1996) method aresubjected to routine sequencing and, if previously unknown (or unknownto be correlated with multidrug-resistance) may be cloned according toconventional molecular engineering techniques as described in CurrentProtocols in Molecular Cloning, Ausubel et al., eds. (1989), GreenePublishing and Wiley Interscience, New York, N.Y. In this manner, theMRP-β probes and/or primers described herein can be used as researchtools to identify and/or produce clones of hitherto unknown genes thatcontribute to multidrug-resistance phenotypes, such as genes thatregulate cellular expression of P-glycoprotein, MRP and/or MRP-β.

The compositions provided as a result of this invention furthermore areuseful tools for the characterization of MRP-β polypeptide structure,biological function and regulation in normal mammalian cells and bodytissues. The availability of information concerning the biological roleof MRP-β is expected to facilitate the design, production and use oftherapeutic agents to treat abnormal phenotypes, particularlydisease-related phenotypes, contributed to by aberrancies in MRP-β. Aspart of this characterization effort, the natural expression pattern ofMRP-β was surveyed in diverse mammalian body tissues. Expressionproducts (total or poly-A(+) RNA) derived from a plurality of human bodytissues were screened for hybridization with a unique MRP-β probefragment as described in EXAMPLE 3. Transport or secretion functionattributable to MRP-β was expected to affect gene expression in cellsand/or tissues responsible for the secretion or excretion of cellularproducts or metabolites. MRP-β was observed to be expressed, at least atlow (detectable baseline) levels, in substantially all body tissues.MRP-β expression similarly can be surveyed in cell types characteristicof a particular body tissue. For this more refined survey, cell typescan be enriched and/or isolated from intact body tissues by conventionmincing, homogenization, collagenase or trypsin digestion procedures,followed by filtration, sedimentation, adherence or panning procedureswell known in the art. Alternatively, cell cultures or cell linesderived from specific body cell types may be used.

Inappropriate alteration of a cellular MRP-β gene, aberrant geneexpression, transcript stabilization, or inappropriate biologicalfunction or stabilization of an MRP-β polypeptide is expected tocorrelate generally with tissue or cell types with a known propensityfor generating transformed cells with inherent or readily acquiredmultidrug-resistance, especially multidrug-resistance that is refractoryto treatment with known chemosensitizing agents or MDR reversal agents.MRP-β production or activity accordingly is likely to fluctuate insecretory epithelial tissues, e.g., respiratory tract, gastrointestinaltract, mammary, urogenital tract, paracrine, endocrine, andneuroendocrine tissues. Sarcomas, carcinomas, especiallyadenocarcinomas, originating from such tissue, particularly thoseoriginating from lung, colon, kidney, bladder, breast, ovarian, uterine,cervical, testicular, prostate or pancreatic tissue, similarly areexpected to inappropriately produce MRP-β and to display or acquiremultidrug-resistance phenotypes. Indeed, confirmation of suchfluctuations already has been obtained, in EXAMPLE 2.

Abnormal or aberrant phenotypes, especially multidrug-resistanceassociated phenotypes, that are contributed to by abnormalitiesaffecting MRP-β, can be treated using pharmaceutical or therapeuticcompositions provided herein. More specifically, the invention providestherapeutic compositions, including prophylactic, palliative andremedial compositions, useful for treatment of any disease state ordeleterious condition contributed to by an abnormality affecting MRP-β.A first category of such therapeutic compositions comprise an antisenseoligonucleotide, or a vector encoding an antisense oligonucleotide, thathybridizes to nucleic acid corresponding to or transcribed from acellular MRP-β gene. Stewart et al. (1996), 51 Biochem, Pharmacol461-469, and Baracchini et al. (1996), U.S. Pat. No. 5,510,239, reportsuccessful, antisense-mediated attenuation of an UP multidrug-resistancephenotype in cultured H69AR cells: exposure to antisenseoligonucleotides significantly reduced intracellular MRP transcript andpolypeptide levels. The techniques and administration methods disclosedtherein can be adapted to provide antisense-mediated attenuation of anMRP-β phenotype as disclosed herein. Stewart et al. (1996) report,however, that attenuation was achieved only transiently, due to the rateof cellular production of new MRP gene transcripts and/or degradation ofthe antisense oligonucleotide. Stewart et al. (1996) notes that, in theadriamycin selected multidrug-resistant H69AR cells, the phenotypecannot be attributed entirely to MRP expression, and for this reasoncounsels that antisense oligonucleotides should be used that arecomplementary to gene regions known to be conserved among members of theABC Transporter Protein family. Similarly, Smyth et al. (1996), PCTPubl. WO 96/02556, reports successful, antisense oligonucleotidemediated, attenuation of a P-glycoprotein based multidrug resistancephenotype in cultured cells wherein the phenotype arises solely fromP-glycoprotein production. By their nature, antisense oligonucleotidesare limited to disruption of their specific target genes. Thus, thedesired result of phenotypic attenuation will not be achieved where themultidrug resistance phenotype arises from (or is preserved by)expression of one or more previously unknown genes, to which theantisense oligonucleotide is unable to hybridize effectively underintracellular conditions.

This limitation is emphasized by the disclosure herein of the presentnovel MRP-β. However, the present disclosure provides basis for thedesign and construction of the present novel antisense oligonucleotides(and oligonucleotide analogs comprising one or more of the modificationsmentioned in Smyth et al. (1996)) competent to hybridize, underintracellular conditions, to all or a unique portion of the MRP-β geneor a transcript thereof. The present antisense oligonucleotides can beused alone or formulated as a cocktail together with one or more of theabove-mentioned antisense oligonucleotides specific to MRP or the MDR-1gene. Antisense oligonucleotides specific for MRP-β can be produced byconventional synthetic or biosynthetic techniques, and formulatedtogether with pharmaceutically acceptable carriers and/or excipientsinto antisense pharmaceutical compositions suitable for local orsystemic administration to an individual, e.g., a cancer sufferer.Suitable pharmaceutical carriers and routes of administration aredescribed in Baracchini et al. (1996), U.S. Pat. No. 5,510,239, andDeeley et al. (1996), U.S. Pat. No. 5,489,519, the teachings of each ofwhich are incorporated herein by reference.

The present MRP-β antisense oligonucleotides accordingly can be used toattenuate any undesirable phenotype associated with MRP-β, such as butnot limited to a multidrug-resistance phenotype attributable in whole orin part to MRP-β expression or overexpression, e.g., in transformedcells in situ in mammalian body tissue. The present antisenseoligonucleotides thus make possible a novel method of potentiatingchemotherapy to eradicate multidrug-resistant transformed cells from thebody of a mammal. The effectiveness of chemotherapy is “potentiated”(enhanced) by restoring or improving vulnerability of the transformedcells to the cytotoxic effects of a chemotherapeutic drug that otherwisewould be ejected from the cell. The method involves administering thedesired chemotherapeutic drug to an individual afflicted with amultidrug-resistant transformed cell population (a tumor, e.g., acarcinoma, sarcoma, leukemia, lymphoma or lymphosarcoma), andcoadministering an above-described antisense pharmaceutical composition.The administration and coadministration steps can be carried outconcurrently or in any order, and can be separated by a time intervalsufficient to allow uptake of either compound by the transformed cellsto be eradicated. For example, the present antisense pharmaceuticalcomposition (or a cocktail composition comprising an MRP-β antisenseoligonucleotide in combination with one or more other antisenseoligonucleotides) can be administered to the individual sufficiently inadvance of administration of the chemotherapeutic drug to allow theantisense composition to permeate the individual's tissues, especiallytissue comprising the transformed cells to be eradicated; to beinternalized by transformed cells; and to disrupt MRP-β gene expressionand/or protein production. The time interval required can be determinedby routine pharmacokinetic means, and should be expected to vary withage, weight, sex, lean tissue content, and health status of theindividual, as well as with size and body compartment location of thepopulation of multidrug-resistant transformed cells to be eradicated.

Similar parameters should be considered in selecting a route ofadministration of the antisense pharmaceutical composition. Thus, thecomposition may be administered locally or systemically, preferably by aparenteral route. The composition can be administered intravenously,intraperitoneally, retroperitoneally, intracisternally, intramuscularly,subcutaneously, topically, intraorbitally, intanasally or by inhalation,optionally in a dispersable or controlled release excipient. One orseveral doses of the present composition may be administered asappropriate to achieve uptake of a sufficient amount of the presentantisense oligonucleotide to produce an attenuation ofmultidrug-resistance phenotype in the transformed cells to be eradicatedby chemotherapy.

The foregoing method alternatively can be accomplished by administrationof a suitable expression vector encoding the present MRP-β antisenseoligonucleotide. Use of the present vector to internally produce oroverproduce the present antisense oligonucleotide is expected toovercome the limitation noted in Stewart et al. (1996), namely, toensure a continuous or renewable level of oligonucleotide mediateddisruption of MRP-β expression or production. In this manner, themultidrug-resistance phenotype can be attenuated, if necessary, for asufficient period of time for the coadministered chemotherapeutic agentto cause the death of transformed cells.

As noted above, all of the foregoing embodiments (MRP-β nucleic acids,host cells, MRP-β protein and antibodies thereto) are useful tocharacterize MRP-β biological function. Natural production of the MRP-βpolypeptide also in untransformed mammalian body tissues likely endowsthe cell with active transport or secretion properties, by which a cellmetabolite, secretion product, or biological response mediator isimported or, more likely, released from the producing cell. Thus, thenormal physiological function of MRP-β may be the transport of one ormore lipids, or substances comprising a moiety with lipid character,across the cell membrane. For example and without being limited byspeculation, MRP-β may transport a bile acid or a steroid hormone orprecursor thereof. Alternatively, MRP-β may mediate cellular uptake ofshort-chain fatty acids as an energy source. Thus, MRP-β may transportnaturally- or synthetically-sourced substances, includingchemotherapeutic drugs, that have salient physical or chemicalproperties in common with the natural transport substrate(s). The MRP-βexpressing host cells provided herein thus are expected to facilitateinvestigation and characterization of substances, including cytotoxins,that are subject to MRP-β mediated transport.

Classical radioassay and/or metabolic radiolabelling techniques can beadapted routinely to screening known cell metabolites and/or secretionproducts to determine which may be a natural MRP-β transportedsubstrate. Phospholipids, glycolipids, extracellular matrix precursors,endocrine hormones, proinflammatory steroids, bile acids, metabolites ofany of the foregoing, and the like can be radiolabeled by incorporationof ³H, ³⁵S, ¹⁴C or ³²P according to standard techniques. Uptake,sequestration and/or efflux of radiolabeled candidate substrates can bemonitored by assessing changes in radioactivity levels (e.g., byscintillation counting, auto-radiography or a similar technique) inMRP-β host cells; in culture medium conditioned by MRP-β host cells; or,as desired, in any appropriate subcellular fraction (e.g., ascintillation fraction) prepared conventionally from MRP-β host cells.Identification of one or more natural substrates for MRP-β may berelevant to the design or selection of potential MRP-β modulators asdescribed below.

Any conventional technique for monitoring cellular susceptibility to acytotoxin of interest, or for monitoring intracellular accumulation,sequestration or efflux thereof, can be adapted with no more thanroutine experimentation to characterization of the biological (e.g.,transport) properties of MRP-β. Thus, the chemosensitivity testing,accumulation and efflux assays summarized in Cole et al. (1994), 54Cancer Res. Cancer Res. 5902-5910 can be used for characterization ofMRP-β export of drugs and/or toxins such as (but not limited to)doxorubicin, vincristine, colchicine, VP-16, vinblastine, verapamil,mitoxantrone, taxol, Cyclosporin A, quinidine, progesterone, tamoxifen,epirubicin, daunorubicin, MX2, and heavy metal ions such as arsenite,arsenate, antimony tartrate, antimonate, and cadmium, whether alone orin any combination thereof. Additional suitable characterization assaysinclude the fluorescence cell sorting techniques disclosed in Krishan(1990), 33 Meth. Cell Biol. 491-500 and in Engel et al. (1996), U.S.Pat. No. 5,556,856 (both incorporated herein by reference), whichcapitalize on the fluorescent properties of daunorubicin. Anothersuitable assay is set forth in Zelle et al. (1996), U.S. Pat. No.5,543,423 (incorporated herein by reference), and is based on assessmentof cellular uptake of vital dyes following a period of exposure to apotentially exportable cytotoxin. Additional published assays aresummarized in Piwnica-Worns (1995), U.S. Pat. No. 5,403,574(incorporated herein by reference), and are based on uptake and/orefflux of fluorescent dyes, such as rhodamine. If desired, the rapidyeast cell-growth monitoring assay set forth in Ruetz et al. (1996), 271J. Biol. Chem. 41544160, also can be applied.

Additional therapeutic methods for treating abnormalities or diseasestates associated with MRP-β, especially with the occurrence of amultidrug-resistance phenotype, are based on the identification and useof modulators, preferably inhibitors, that affect MRP-β gene activationor expression, transcript stability, polypeptide production,post-translational processing, insertion into cellular phospholipidmembranes, stabilization and/or biological function, especiallytransport function. A candidate substance that detectably affects(products a fluctuation in) any of the foregoing MRP-β parameters isidentified herein as an MRP-β modulator. Thus, for example, a candidatethat interferes with host cell resistance to a cytotoxin is identifiedherein as a preferred inhibitory modulator (inhibitor) of MRP-β.Candidate sustances to be subjected to screening and/or identificationmethods described herein available or can be produced by routineadaptations of teachings set forth in Intelligent Drug Design, A NatureSupplement, 384 Nature, Suppl. to No. 6604 (1996). Additional exemplarysources of candidate MRP modulators are taught in Agrafiotis et al.(1995), U.S. Pat. No. 5,463,564; Zambias et al. (1996), PCT Publ. No.WO96/22529; Hogan et al. (1996), PCT Publ. No. WO96/12482; Hogan (1995),PCT Publ. Nos. WO95/32184 and WO95/18972; and, Beutel et al. (1995), PCTPubl. No. WO95/27072. Preferred candidate substances are smallmolecules, e.g., elements of a combinatorial chemistry or naturalproducts library or pharmacopoeia. Currently, multidrug-resistantderivatives of the MCF-7 cell line, or MCF-7 host cells displaying avector-derived, cell surface MRP-β polypeptide, are preferred herein forthe identification of modulators of MRP-β. Any of the above-mentionedassays can be used for the present purpose, including high-throughputcell survival assays that monitor whether the present MRP-β expressingMCF-7 cells survive exposure to cytotoxin levels at which non-resistantcells normally succumb. For example, survival of MRP-β expressing hostcells can be compared to survival of mock transfected MCF-7 cells atequivalent cytotoxin concentrations.

As mentioned previously herein, several inhibitors or antagonists of theknown mammalian ABC Transporter Proteins, P-glycoprotein and MRP, havebeen disclosed. An inhibitor or antagonist that achieves completeinterference with gene expression, polypeptide production and/orfunction effectively reverses the multidrug resistance phenotype,restoring cellular vulnerability to the cytotoxic effects of anotherwise exported chemotherapeutic drug. An inhibitor or antagonistthat achieves partial interference also can be considered beneficialclinically, in that partial interference with drug export function“attenuates” or reduces penetrance of the multidrug resistancephenotype. Upon treatment with a partial inhibitor, cellularvulnerability to cytotoxins is increased, albeit not fully restored.Such substances are commonly referred to in the art as “MDR reversalagents” or “chemosensitizing agents.” Powell et al. (1996), U.S. Pat.Nos. 5,561,141 and 5,550,149; Powell et al. (1995), U.S. Pat. No.5,387,685; Engel et al. (1996), U.S. Pat. No. 5,556,856; Zelle et al.(1996), U.S. Pat. No. 5,543,423; Sunkara (1996), U.S. Pat. No.5,523,304; Sunkara et al (1993), U.S. Pat. Nos. 5,190,957 and 5,182,293;Sarkadi et al. (1995), PCT Publ. WO 95/31474; Piwnica-Worms (1995), U.S.Pat. No. 5,403,574; Hait et al. (1992), U.S. Pat. No. 5,104,856. Littlestructural similarity has been observed between the known classes of MDRreversal agents, or between reversal agents and exported cytotoxicdrugs. Thus, high through-put screening, e.g., of naturally-sourced orsynthetic chemicals in a pharmacopoeia or combinatorial library, wasrequired to identify each currently known MDR reversal agent.Furthermore, the majority of known MDR reversal agents are specificinhibitors of either P-glycoprotein or of MRP: little to nocross-inhibition has been observed. Thus, it is expected that empiricalscreening will be required, for the identification of one or moremodulators, preferably inhibitors, of MRP-β. Exemplary identification orscreening protocols are referenced herein and appear herein in EXAMPLES4 and 5.

All modulators of MRP-β, including partial modulators, that areidentified through practice of the above-described methods, or routinemodifications thereof, are considered to be within the scope of thepresent invention. Small molecule modulators are preferred. Inhibitorymodulators (inhibitors) are especially contemplated herein. Fortherapeutic administration purposes, a modulator of the presentinvention can be administered to an individual as a pharmaceuticallyacceptable salt or derivative. Further, the present modulator can beformulated with any pharmaceutically acceptable carrier, excipient,adjuvant or vehicle. Appropriate pharmaceutically acceptable salts,derivatives, carriers, excipients, adjuvants and vehicles are asdisclosed in Zelle et al. (1996), U.S. Pat. No. 5,543,423 (which isincorporated herein by reference) or can be produced or selected byroutine modifications thereof.

The present MRP-β modulator accordingly can be used to mitigate severityof, up to an including to abrogate, any phenotype associated with anabnormality affecting MRP-β. That is, the present modulator may be usedto treat or palliate any disease or condition affecting the healthstatus of an individual, such as a human, that arises from the MRP-βabnormality. The modulator also may be administered prophylactically, toavert or delay the onset of a deleterious phenotype associated withMRP-β dysfunction. In particular, the present NW-0 modulator is usefulto attenuate a multidrug-resistance phenotype attributable in whole orin part to MRP-β gene abnormality, gene expression, transcriptstabilization, or polypeptide production, processing, stability orbiological function, e.g., in transformed cells in situ in mammalianbody tissue. Preferred inhibitory modulators make possible novelmethods, for example, of potentiating chemotherapy to eradicatemultidrug-resistant transformed cells from the body of a mammal. As withthe antisense pharmaceutical composition method discussed herein, theeffectiveness of chemotherapy is enhanced by restoring or improvingvulnerability of the transformed cells to the cytotoxic effects of achemotherapeutic drug that otherwise would be ejected from the cell. Thepresent, modulator-based method involves administering the modulatoralone or as an adjuvant to the desired chemotherapeutic drug, to anindividual afflicted with a multidrug-resistant tumor, e.g., acarcinoma, sarcoma, leukemia, lymphoma or lymphosarcoma. Thechemotherapeutic drug and MRP-β modulator may be administeredconcurrently or in any order, and can be separated by a time intervalsufficient to allow uptake of either compound by the transformed cellsto be eradicated. If desired, the present modulator can be administeredalone or in a cocktail, combined with one or more known MDR reversalagents (e.g., agents that affect MRP or P-glycoprotein).

Preferably, the modulator is administered to the individual sufficientlyin advance of administration of the chemotherapeutic drug to allow themodulator to permeate the individual's tissues, especially tissuecomprising the transformed cells to be eradicated; to be internalized bytransformed cells; and to impair MRP-β mediated cytotoxin sequestrationor efflux. The time interval required can be determined by routinepharmacokinetic means, and should be expected to vary with age, weight,sex, lean tissue content, and health status of the individual, as wellas with size and body compartment location of the population ofmultidrug-resistant transformed cells to be eradicated. Similarparameters should be considered in selecting a route of administrationof the modulator. Thus, the modulator may be administered locally orsystemically, preferably by a parenteral route. It can be administeredintravenously, intraperitoneally, retroperitoneally, intracisternally,intramuscularly, subcutaneously, topically, intraorbitally, intranasallyor by inhalation, optionally in a dispersable or controlled releaseexcipient. One or several doses may be administered as appropriate toachieve uptake of a sufficient amount of the present modulator toproduce an attenuation of multidrug-resistance phenotype in thetransformed cells to be eradicated by chemotherapy. As a result oftherapeutic intervention with an MRP-β modulator (preferably, aninhibitory modulator), penetrance of an abnormal or deleteriousphenotype (generally, but not limited to, a multidrug resistancephenotype) is attenuated, even abrogated, in the treated individual. Theoverall dosage and administration protocol for treatment with thepresent modulator may be designed and optimized by the clinicalpractitioner through the application of routine clinical skill.

Practice of the invention will be still more fully understood from thefollowing EXAMPLES, which are presented solely to illustrate principlesand operation of the invention, and should not be construed as limitingscope of the invention in any way.

EXAMPLE 1 Isolation and cloning of fill-length MRP-β cDNA

A unique fragment (SEQ ID No: 3) of the novel MRP-β gene was identifiedby computer-assisted screening of a nucleic acid database correspondingto a human endothelial cell expression library. The library was preparedusing cellular RNA transcripts produced in human microvascularendothelial cells (HUMVEC) isolated from breast tissue and maintained inprimary culture in the presence of a commercially availableextracellular matrix composition (Matrigel), and in the presence ofappropriate growth and differentiation factors (e.g., vascularendothelial cell growth factor (VEGF)). These conditions had previouslybeen shown to preserve cell viability and substantially differentiatedphenotype in vitro.

A nucleic acid probe corresponding to the SEQ ID No: 3 unique fragmentwas prepared by conventional techniques. This probe was used forhybridization screening of the HUMVEC expression library for thepresence of MRP-β cDNAs. This procedure yielded an MRP-β cDNA (residues67-4847 FIG. 1A-G and SEQ ID No: 1), 4.78 kb (kilobases) in length. Theclone comprising this cDNA insert has been designated fohd013a05m anddeposited with the American Type Culture Collection. Two independentcDNA clones comprising approximately 60 residues upstream (5′) from thefohd013a05m MRP-β insert were isolated by hybridization screening ofhuman brain and liver cDNA libraries with a nucleic acid probecorresponding approximately to the 5′ 0.5 kb of the fohd013a05m MRP-βinsert. This probe was prepared by isolating an approximately 0.5 kbSacI fragment from fohd013a05m. The cDNA sequence presented in SEQ IDNo: 1 comprises the sequence of the fohd013a05m MRP-β insert and thesequence of an additional 66 upstream (5′) nucleotides. The open readingframe (ORF) of the SEQ ID No: 1 cDNA encodes an MRP-β polypeptide (SEQID No: 2) 1437 amino acid residues in length and in addition, includes a0.42 kb 3′ untranslated region. The ORF start site indicated in SEQ IDNo: 1 (at nucleotides 116-118 of SEQ ID No: I) is the first in-frame ATGcodon downstream from the TGA stop codon at nucleotides 23-25 of SEQ IDNo: 1.

EXAMPLE 2 Correlation of MRP-β expression level withmultidrug-resistance phenotype.

Involvement of the present novel MRP-β gene in the acquisition ormaintenance of a multidrug-resistance phenotype has been confirmed bycomparing the level of MRP-β gene expression in immortalized,transformed cells (wild-type or parent cells) that have not acquired theproperty of multidrug-resistance with the level in a multidrug-resistantderivative of the parent cell population. One set of exemplary parentand multidrug-resistant derivative cell lines are described in Mirsky etal. (1987), 47 Cancer Res. 2594-2598 (parent and multidrug-resistant(MDR) derivative of the H69 human small cell lung carcinoma line).Additional exemplary parent and multidrug-resistant derivative lines aredescribed in Slapak et al. (1994), 84 Bloo 3113-3121 (parent and MDRderivative of the U937 human myeloid leukemia line); Batist et al.(1986), 261 J. Biol. Chem. 15544-15549 (parent and MDR derivative of theMCF-7 human breast adenocarciinoma line); March et al. (1986), 46 CancerRes. 4053-4057 (parent and MDR derivative of the HL-60 humanpromyelocytic leukemia line); and, Hamilton et al. (1984), 11 Sem.Oncol. 285-298 (parent and MDR derivative of the A2780 human ovariancarcinoma line). Each of the foregoing references is incorporated hereinby reference. To demonstrate correlation between MRP-β gene expressionand multidrug-resistance phenotype, parental (wild-type) andadriamycin-selected multidrug resistant MCF-7 cells were cultured toconfluency under standard cell culture conditions and treated to releaseexpressed nucleic acid transcripts, which were subjected to Northernblot analysis.

Preparation of cellular RNA. Expressed nucleic acids were isolated fromthe exemplary parental and resistant MCF-7 cells using components of theQiagen, Inc. RNeasy Total RNA kit, generally as in the Qiagen, Inc.RNeasy Handbook (1995). Kit components include spin columns, collectiontubes, lysis buffer, wash buffer and RNAse free water. Expressed nucleicacid extracts were prepared by suspending cells in lysis buffersupplemented with 2-mercaptoethanol and passage of the resulting mixturethrough a Qiagen, Inc. Qiashredder homogenization column. RNA waspurified from the resulting lysate using a Qiagen, Inc. RNeasy columnsupplied with the kit. The lysate was loaded onto the RNeasy colum,washed and RNA was eluted generally as described in the RNeasy Handbook.

Electrophoretic Resolution of Expressed RNAs. Agarose-formaldehyde slabgels (1.0-2.5% agarose) were prepared and cast according to standardtechniques. RNA samples (10-30 μg total RNA or 1-3 μg PolyA(+) RNA) werecombined with denaturing bromophenol blue sample buffer, loaded onto thegel and subjected to electrophoresis by passage of 100 volts through thegel chamber for about 3 hours or until the bromophenol blue dye fronthad migrated about 10 cm into the gel. A photograph of the resolved gelwas obtained prior to transfer of resolved RNAs to nylon.

Replica Transfer of Resolved RNAs to Nylon. The gel comprising resolvedcellular MRP-β transcript was prepared for transfer by soaking in 0.05 NNaOH, 0.15 M NaCl for 20-30 minutes, followed by neutralization in 0.1 MTris pH 7.5, 0.15 M NaCl for 30 minutes. RNA contents of theneutralization gel were then transferred to a nylon membrane using aPosiblot apparatus (Stratagene, Inc.). Transfer was allowed to proceedfor 1 hour, following which the transferred, resolved RNAs werecrosslinked to the membrane using UV light generated by a Stratalinkerapparatus (Stratagene, Inc.). The location of resolved RNAs on themembrane was visualized by staining with methylene blue. The positionsof the RNA ladder, 18S, and 28S ribosomal RNAs were marked on aphotograph taken of the stained membrane, which was then destainedaccording to standard procedure.

Preparation of detectably labeled MRP-β Probe. A unique fragment (e.g.,SEQ ID No: 3) of the MRP-β cDNA was used for the preparation of aradiolabeled hybridization probe for visualizing the electrophoreticallyresolved, full-length MRP-βtranscript expressed in parent (wild-type)and MDR MCF-7 cells. The probe was prepared using the Stratagene, Inc.Prime It-RmT Primer Labeling Kit, generally according to the protocolsupplied by the manufacturer (see also Feinberg et al. (1984) 137 Anal.Biochem. 266-267 and Feinberg et al. (1983), 132 Anal. Biochem. 6-13).Kit components include control DNA, Magenta thermostable DNA polymerase,stop mix, and dehydrated single-use reaction mixtures comprising randomprimers, nucleotides, buffer and cofactors required by the polymerase.To prepare the probe, 50 ng MRP-βDNA (e.g., cDNA insert comprising SEQID No: 3) in aqueous solution was added to a kit single-use reactionmixture and boiled to ensure denaturation. To obtain incorporation of atleast 10⁶ cpm/μL, 5 μL [α-³²P]dCTP (6000 Ci/mmol) was added to themixture, followed by 3 mL Magenta polymerase (4U/uL). Probe synthesiswas conducted at 37° C. for 10 minutes, then stopped by the addition of2 μL stop mix. To reduce background, the labeled probe was purifiedusing a chromaspin TE-10 column prior to use for hybridization.

Hybridization. Prior to contact with the radiolabeled probe, the nylonmembrane comprising crosslinked, electrophoretically resolved MCF-7cellular transcripts was prehybridized for 20 minutes at 65° C. in 10 mLRapid-hyb solution available from Amersham, Inc. The prepared probe wasboiled for 5 minutes to ensure denaturation, and added to an additional10 mL Rapid-Hyb solution. The prehybridization solution was exchangedfor probe solution, and the probe was allowed to hybridize tomembrane-bound transcripts for 2 hours at 65° C. Excess, unhybridizedprobe was removed by washing the membrane in 2×SSC, 0.1% SDS for 20minutes, either at room temperature or at 42° C. Thereafter, themembrane was washed in 0.1×SSC, 0.1% SDS for 20 min. at 65° C. The 65°C. wash step was repeated if necessary to obtain a satisfactorysignal-to-background ratio as assessed by geiger counter. Results werevisualized by exposure to X-ray film according to standard procedures.Thereafter, MRP-β probe was stripped by addition of a boiling solutionof 0.5% (w/v) SDS (0.1×SSC, 0.1% SDS also can be used as a strippingsolution). Significance of the MRP-0 results were verified byrehybridization of the membrane with a probe specific for the transcriptof a conventionally used housekeeping or structural gene (e.g., Ef-TU oractin).

Results. A single 6 kb transcript was visualized by the MRP-β probe inboth wild-type and MDR MCF-7 cellular RNA. A significantly elevatedlevel of the MRP-β transcript was observed in the MDR derivative cellline, which is reported in Batist et al. (1986) to be 192-fold moreresistant to adriamycin than the parental (wild-type) MCF-7 human breastadenocarcinoma cell line. Consistent results showing elevated levels ofMRP-β gene expression were observed in comparison studies of parentaland MDR derivative cell lines established from human ovarian carcinoma(A2780; Hamilton et al. (1984)) and human leukemias (HL-60; March et al.(1986), and U937; Slapak et al. (1994)). Thus, MRP-β gene expressionlevel correlates with the acquisition of a multidrug-resistancephenotype, rather than with the body tissue type in which a particulartumor arises.

EXAMPLE 3 Expression of MRP-β in Mammalian Body Tissues

As noted above, a clearly detectable baseline level of MRP-β geneexpression was observed even in wild-type tumor cell lines. To establishwhether this baseline expression correlates with tumorigenesis, theabove-described radiolabeled MRP-βprobe was hybridized to commerciallyavailable human multiple tissue Northern (MTN) blots (Clontech, Inc.),generally according to the manufacturer's directions and the proceduredescribed above in EXAMPLE 2. Tissues from which polyA(+)RNA wasanalyzed included heart, brain, placenta, lung, liver, skeletal muscle,kidney, pancreas, spleen, thymus, prostate, testis, ovary, smallintestine, colon (mucosal lining) and peripheral blood leukocyte.

Results. Clearly detectable baseline expression of a 6 kb MRP-βtranscript was observed in substantially all normal human body tissuessurveyed, with the highest expression level being observed in hearttissue. The survey samples represent expressed RNAs isolated fromlysates of whole tissue, rather than from specific cell typescharacteristic of one or more body tissues. Taken together with theisolation of MRP-β cDNAs from a HUMVEC expression library (described inEXAMPLE 1), the present MTN survey data is consistent with substantiallyubiquitous baseline expression of MRP-β in vasculature ormicrovasculature.

EXAMPLE 4 Confirmation that MRP-β expression is sufficient to confer asurvival advantage on cells exposed to a cytotoxic agent

Host cells stably transfected with an MRP-β expression vector asdescribed herein are expected to gain a significant survival advantage,relative to source (untransfected) or control (antisense transfected)cells. To establish this survival advantage, triplicate cultures ofMRP-β host cells, control cells and source cells (e.g., MCF-7 humanbreast adenocarcinoma cells) are generated in 24-well cell cultureplates. Once the cultures have attained at least 80% confluency, lethalor sub-lethal amounts of a cytotoxin (e.g., adriamycin, bisantrene) areadded to each well. After a sufficient period of time for cytotoxiceffects to be manifested (e.g., 16-24 hours in culture), culture mediacomprising the cytotoxic drug are aspirated or otherwise removed, andcells are stained with a vital dye such as Trypan blue. Whichcommercially available vital dye is used in this procedure is a matterof choice; thus, sulforodoamine B (see Powell et al. (1990), U.S. Pat.No. 5,550,149) could be used in lieu of Trypan blue. The number of cellsthat remain viable (e.g., capable of excluding the dye) are countedusing a hemocytometer, flow cytometer or other appropriate device.

Expected Results, MRP-β expressing host cells are expected to acquirethe capability of surviving exposure to otherwise lethal amounts of acytotoxin, such as adriamycin or bisantrene. Analysis of thedifferential between toxin levels that are lethal to source or controlcells, and that which is lethal to MRP-β host cells, is expected toprovide a predictive index of the recalcitrance of MRP-β expressingtransformed cells in situ to chemotherapy. Repetition of thiscytotoxicity assay with additional toxins (e.g., environmentally oroccupationally derived toxins, metabolites or chemotherapeutic drugs) isexpected to elucidate the nature of substances exportable orsequestrable by MRP-β and to uncover specific differences between thecharacteristics of substrates transported by MRP-β and those transportedby known ATP Transporter Protein superfamily members such asP-glycoprotein and/or MRP.

Screening for a modulator of MRP-β. The present cytotoxicity assay canbe adapted routinely to provide a rapid assay for screening candidatemodulators of MRP-β. In this adaptation, host cell cultures areincubated in the presence of a toxin to which MRP-β expression confers asurvival advantage. The level of toxin exposure is sub-lethal to hostcells but lethal to source cells or control cells. Candidate MRP-βmodulators (e.g., inhibitors) are added to the cell cultures, which areincubated for a sufficiently further period of time for cytotoxicity tobe manifested (e.g., 16-24 hours). A candidate that attenuates orabrogates the host cells' survival advantage is identified as an MRP-βinhibitor. Guidelines for this adaptation of the present cytotoxicityassay may be found in Powell et al. (1996), U.S. Pat. No. 5,550,149.Candidate MRP-β modulators may be selected from any appropriate source,such as a pharmacopeia of natural or synthetic substances, combinatorialchemistry library, phage display epitope library, or the like.Appropriate sources are available or can be produced by routineadaptations of teachings set forth in Intelligent Drug Design, A NatureSupplemenL 384 Nature, Suppl. to No. 6604 (1996). Additional exemplarysources of candidate MRP-β modulators are taught in Agrafiotis et al.(1995), U.S. Pat. No. 5,463,564; Zambias et al. (1996), PCT Publ. No.WO96/22529; Hogan et al. (1996), PCT Publ. No. WO96/12482; Hogan (1995),PCT Publ. Nos. WO95/32184 and WO95/18972; and, Beutel et al. (1995), PCTPubl. No. WO95/27072.

EXAMPLE 5 Assessment of MRP-β Mediated Drug Efflux

Without being limited by speculation, it is likely that MRP-β confersthe above-described survival advantage by mediating sequestration orefflux of one or more cytotoxins. That is, it is likely that MRP-β is amember of the ABC Transporter Protein superfamily that carries out anexport function. However, routine empirical testing is required toconfirm whether MRP-β exports one or more toxic substances, or importsone or more nutrients or energy sources, such as sugars or fatty acidsof dietary or other metabolic origin. A number of conventional protocolscan be practiced, with such routine modifications as may be deemedappropriate by the practitioner, to establish whether MRP-β mediatestoxin export. A presently preferred technique capitalizes on thefluorescent properties of anthracycline toxins (including adriamycin(doxorubicin) and daunamycin), such that toxin accumulation and/orefflux from MRP-β expressing host cells can be monitored by fluorescencehistochemistry or, preferably, by fluorescence-activated flow cytometry.An example of this technique is described in Krishan (1990), 33 Meth.Cell Biol, 491-500, incorporated herein by reference.

Fluorescent labeling. Viable MRP-β host cells (at least 10,000) aresuspended in culture medium in the sampling cuvette of a flow cytometer,such as the EPICS 753 apparatus (Coulter Electronics, Inc.) equippedwith an argon laser for fluorophore excitation at 488 nm, and aphotomultiplier (e.g., MDADS II data acquisition apparatus) fordetection of 530 nm emissions. The cuvette is maintained at 37° C., andadriamycin or daunomycin are added to a final concentration of 1-3 μMprior to cell sorting. Two-parameter histograms are generated based oncellular fluorescence and incubation time (typically 30 to 60 minutes)in the presence of the fluorescent toxin.

Expected results. MRP-β host cells are expected to internalize and/orretain significantly lower levels of adriamycin or daunomycin thansource cells or control cells.

Screening for a modulator of MRP-β. The above drug efflux assay can beadapted routinely to provide a rapid assay for screening candidatemodulators of MRP-β. In this adaptation, a candidate MRP-β modulator isadded to the cuvette during the fluorophore uptake incubation. Acandidate that attenuates or abrogates the host cells' capacity forfluorophore efflux is identified as an MRP-β inhibitor. Guidelines forthis adaptation may be found in Krishan (1990), 33 Meth. Cell Biol.491-500). Candidate MRP-β modulators may be selected from anyappropriate source, such as the sources mentioned in EXAMPLE 4.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. For example, theinvention may be embodied in one or more variants, e.g., deletion,addition or substitution variants, of the nucleic acid and/or proteinsequences disclosed herein, such as may be produced routinely bymutagenesis or other conventional molecular engineering and biosyntheticproduction techniques. Specifically, the invention may be embodied inany variant, whether biosynthetically produced or isolated from anatural source, the expression or overexpression of which endows amammalian cell with a multidrug-resistance phenotype. More specifically,the invention may be embodied in a variant which, when expressed oroverexpressed, endows a mammalian cell with resistance to the cytotoxiceffects of MRP-β transportable drugs. The foregoing embodiments aretherefore to be considered in all respects illustrative rather thanlimiting on the invention described herein. Scope of the invention isthus indicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are intended to be embraced therein.

1. A method of identifying an inhibitor of MRP-β, comprising the stepsof: (a) contacting a cell with a candidate inhibitor of MRP-β; (b)assaying the level of expression of the MRP-β nucleic acid molecule setforth as SEQ ID No: 1 in said cell, wherein a detectable decrease insaid level indicates that said candidate modulator inhibitor is an MRP-βinhibitor, thereby identifying an MRP-β inhibitor.
 2. A method ofidentifying an inhibitor of MRP-β, comprising the steps of: (a)contacting a cell with a substrate exported or sequestered by MRP-β,said cell expressing a vector-derived MRP-β polypeptide, the amino acidsequence of which shares at least 90% sequence identity with SEQ ID No:2, wherein said MRP-β functions to transport, expel, or sequestersubstances from an intracellular milieu, and wherein; (b) contactingsaid cell with a candidate modulator inhibitor of MRP-β; (c) assayingfor a detectable decrease in export or sequestration of said substrate,wherein a detectable decrease in said export or sequestration indicatesthat said candidate inhibitor is an MRP-β m inhibitor, therebyidentifying an MRP-β inhibitor.
 3. A method of identifying a inhibitorof MRP-β, comprising the steps of: (a) contacting a cell with acytotoxin exported or sequestered by MRP-β, said cell expressing avector-derived MRP-β polypeptide, the amino acid sequence of whichshares at least 90% sequence identity with SEQ ID No: 2, wherein saidMRP-β functions to transport, expel, or sequester substances from anintracellular milieu; (b) contacting said cell with a candidatemodulator inhibitor of MRP-β; (c) assaying survival of said cell,wherein a detectable decrease in said survival indicates that saidcandidate inhibitor is an MRP-β inhibitor, thereby identifying an MRP-βinhibitor.
 4. A method of identifying an inhibitor of MRP-β, comprisingthe steps of: (a) contacting a cell with a candidate inhibitor; (b)assaying the level of expression of the MRP-β polypeptide set forth asSEQ ID No: 2 in said cell wherein said MRP-β functions to transport,expel, or sequester substances from an intracellular milieu, wherein adetectable decrease in said level indicates that said candidateinhibitor is an MRP-β inhibitor, thereby identifying an MRP-β inhibitor.5. A method of identifying an inhibitor of MRP-β, comprising the stepsof: (a) contacting a cell with a substrate exported or sequestered byWP-0, said cell expressing a vector-derived MRP-β polypeptide encoded bya nucleic acid molecule which hybridizes under conditions ofhybridization in 0.5M NaHPO₄ at 65° C. followed by washing in 0.1×SSC at68° C. to a complement of the nucleic acid molecule having the sequenceof SEQ ID No: 1, wherein said MRP-β polypeptide functions to transport,expel, or sequester substances from an intracellular milieu; (b)contacting said cell with a candidate inhibitor of MRP-β; (c) assayingfor a detectable decrease in export or sequestration of said substrate,wherein a detectable decrease in said export or sequestration indicatesthat said candidate inhibitor is an MRP-β inhibitor, thereby identifyingan MRP-β inhibitor.
 6. A method of identifying an inhibitor of MRP-β,comprising the steps of: (a) contacting a cell with a substrate exportedor sequestered by MRP-β, said cell expressing a vector-derived MRP-βpolypeptide encoded the nucleic acid molecule having the sequence of SEQID No: 1; (b) contacting said cell with a candidate modulator inhibitorof MRP-β; (c) assaying for a detectable decrease in export orsequestration of said substrate, wherein a detectable decrease in saidexport or sequestration indicates that said candidate inhibitor is anMRP-β inhibitor, thereby identifying an MRP-β inhibitor.
 7. A method ofidentifying an inhibitor of MRP-β, comprising the steps of: (a)contacting a cell with a substrate exported or sequestered by MRP-β,said cell expressing a vector-derived MRP-β polypeptide by the DNAinsert of the plasmid deposited as ATCC Deposit No. 94809; (b)contacting said cell with a candidate inhibitor of MRP-β; (c) assayingfor a detectable decrease in export or sequestration of said substrate,wherein a detectable decrease in said export or sequestration indicatesthat said candidate inhibitor is an MRP-β inhibitor, thereby identifyingan MRP-β inhibitor.
 8. A method of identifying an inhibitor of MRP-β,comprising the steps of: (a) contacting a cell with a cytotoxin exportedor sequestered by MRP-β said cell is expressing a vector-derived MRP-βpolypeptide encoded by a nucleic acid molecule which hybridizes underconditions of hybridization in 0.5M NaHPO₄ at 65° C. followed by washingin 0.1×SSC at 68° C. to a complement of the nucleic acid molecule havingthe sequence of SEQ ID No: 1, wherein said MRP-(3 functions totransport, expel, or sequester substances from an intracellular milieu,and wherein; (b) contacting said cell with a candidate inhibitor ofMRP-β; (c) assaying survival of said cell, wherein a detectable decreasein said survival indicates that said candidate inhibitor is an MRP-βinhibitor, thereby identifying an MRP-β inhibitor.
 9. A method ofidentifying an inhibitor of MRP-β, comprising the steps of: (a)contacting a cell with a cytotoxin exported or sequestered by MRP-β saidcell expressing a vector-derived MRP-D polypeptide encoded the nucleicacid molecule having the sequence of SEQ ID No: 1; (b) contacting saidcell with a candidate inhibitor of MRP-β; (c) assaying survival of saidcell, wherein a detectable decrease in said survival indicates that saidcandidate inhibitor is an MRP-β inhibitor, thereby identifying anMRP-βinhibitor.
 10. A method of identifying an inhibitor of MRP-β,comprising the steps of: (a) contacting a cell with a cytotoxin exportedor sequestered by MRP-β, said cell expressing a vector-derived MRP-βpolypeptide by the DNA insert of the plasmid deposited as ATCC DepositNo. 94809; (b) contacting said cell with a candidate inhibitor of MRP-β;(c) assaying survival of said cell, wherein a detectable decrease insaid survival indicates that said candidate inhibitor is an MRP-βinhibitor, thereby identifying an MRP-β inhibitor.
 11. The method ofclaim 2 or claim 3, wherein the amino acid sequence of thevector-derived MRP-β polypeptide shares at least 95% sequence identitywith the amino acid sequence of SEQ ID No:
 2. 12. The method of any oneof claims 2 and 5-7, wherein the substrate is a cytotoxin.
 13. Themethod of any one of claims 2 3 and 5-10 wherein MRP-β expressionconfers a survival advantage on said cell.
 14. The method of any one ofclaims 2-3 and 5-10, wherein the cell expresses a cell surface MRP-βpolypeptide.
 15. The method of any one of claims 1-3 and 5-10, whereinthe cell is a eukaryotic cell.
 16. The method of any one of claims 1-3and 5-10, wherein the cell is a yeast or mammalian cell.
 17. The methodof any one of claims 1-3 and 5-10, wherein the cell is a human cell. 18.The method of any one of claims 1-3 and 5-10, wherein the cell is aMCF-7 cell.
 19. The method of claim 1, wherein assaying the level ofMRP-β comprises assaying the amount or rate of production of MRP-βnucleic acid molecule.
 20. The method of claim 4, wherein assaying thelevel of MRP-β comprises assaying the amount or rate of production ofMRP-β polypeptide is in said cell.
 21. The method of any one of claims1-3 and 5-10, wherein the candidate inhibitor is contacted with the cellprior to, concomitantly with, or following exposure to the substrate.22. The method of any one of claims 1-3, wherein the candidate inhibitoris selected from the group consisting of a natural metabolite, asynthetic chemical, a synthetic metabolite, a toxin, an antibiotics, anelement of a combinatorial chemistry library, an element of a nucleotidelibrary, an element of a peptide library, a naturally sourced chemical,a naturally sourced cell secretion product, a cell lysate.
 23. Themethod of any one of claims 1-3, wherein the candidate inhibitor is asmall molecule.
 24. The method of claim 2 or 3, wherein the amino acidsequence of the vector-derived MRP-β polypeptide comprises the aminoacid sequence of SEQ ID No: 2.